Application of a yam exosome in preparation of a medicine for preventing colitis

Abstract

This invention discloses the application of yam exosomes in the preparation of drugs for the prevention of colitis, belonging to the field of plant exosome technology. This invention employs a polyethylene glycol (PEG) precipitation method combined with ultracentrifugation to extract yam exosomes. Compared with the traditional sucrose density gradient centrifugation method, this method has a simpler operation process and can efficiently obtain yam exosomes with uniform morphology and intact structure. The obtained yam exosomes can not only prevent colitis but also effectively improve loperamide-induced constipation symptoms.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of yam exosomes in the preparation of drugs for the prevention of colitis. Background Technology

[0002] The main pathological feature of ulcerative colitis (UC) is the disruption of the intestinal mucosal barrier, leading to bacterial translocation and persistent inflammation. Restoring the integrity of the intestinal mucosal barrier is key to the treatment of UC. Existing drugs, such as mesalazine, primarily focus on anti-inflammation, with limited direct repair effects on the physical mucosal barrier.

[0003] Plant exosomes (PENs), as a novel nanomedicine carrier, have shown promising bioactivity in recent years. However, exosomes from different plant sources have varying functional focuses due to differences in their endogenous components. For example, ginger exosomes focus on anti-oxidation, while grape exosomes focus on anti-apoptosis.

[0004] Yam (Dioscorea polystachya) is a traditional food and medicine variety. Traditional Chinese medicine believes it has the effects of "strengthening the spleen and stomach, nourishing the kidneys and replenishing essence." Modern research shows that it is rich in yam polysaccharides and mucoproteins, which have a protective effect on the gastrointestinal tract. However, directly extracting exosomes from yam faces significant technical challenges: yam tubers contain a large amount of high-molecular-weight mucoproteins and starch, which easily form a network structure to encapsulate exosomes, resulting in extremely low extraction rates using conventional differential centrifugation. Furthermore, the product contains a large amount of free polysaccharides, interfering with efficacy evaluation. Currently, there are no reports on high-purity yam exosomes and their application in colitis. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an application of yam exosomes in the preparation of drugs for the prevention of colitis.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of yam exosomes in the preparation of drugs for the prevention of colitis, characterized in that: The drug contains an effective dose of yam exosomes; The yam exosomes have at least one of the following effects: a. Significantly repairs damaged intestinal mucosal barrier; b. Reduces the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in colon tissue; c. Regulate the expression of gastrointestinal hormones, including reducing VIP and ET-1 expression and increasing MTL and SP expression. d. Improves intestinal excretion function, including increasing stool weight and water content, shortening the time to the first blue stool, improving small intestinal propulsion rate, and increasing the number of stool particles within 6 hours.

[0009] In a preferred embodiment of the application described in this invention, the mass percentage of yam exosomes in the drug is 2% to 15%.

[0010] In a preferred embodiment of the application described in this invention, the dosage of the drug is 20-60 mg / kg / day, and the route of administration is oral gavage or rectal administration.

[0011] In a preferred embodiment of the application described in this invention, the dosage form of the drug is a retention enema solution, enteric microcapsules, nanogels, or lyophilized powder.

[0012] As a preferred embodiment of the application described in this invention, the method for extracting yam exosomes includes the following steps: Wash, peel, and chop the fresh yam raw material. Mix it with PBS buffer at 4℃ and blend it into a paste. Filter the paste and collect the filtrate. Centrifuge the obtained filtrate at differential speed, discard the precipitate, and collect the supernatant; Take the supernatant and centrifuge it at ultraspeed to collect the supernatant; The supernatant after ultracentrifugation was mixed with 24% PEG6000 and allowed to stand at 4°C for 24 hours. After standing, the resulting mixture was centrifuged at 4℃, 10000rpm for 1h, and the precipitate was collected. The precipitate was yam exosomes.

[0013] As a preferred embodiment of the application described in this invention, the differential centrifugation is performed under the following conditions: centrifugation is performed three times at 4°C, with the centrifugation conditions being 2000 rpm, 10 min, 6000 rpm, 30 min, and 10000 rpm, 40 min in sequence.

[0014] In a preferred embodiment of the application described in this invention, the mass ratio of the PBS buffer to the yam is 2:1.

[0015] In a preferred embodiment of the application described in this invention, the volume ratio of the supernatant after ultracentrifugation to PEG6000 is 1:1.

[0016] In a preferred embodiment of the application described in this invention, the colitis is ulcerative colitis, radiation enteritis, or intestinal mucosal damage induced by chemotherapy drugs.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical composition for repairing the intestinal mucosal barrier, characterized in that it comprises yam exosomes prepared by any one of claims 5 to 8, and a pharmaceutically acceptable carrier.

[0018] Beneficial effects of this invention: (1) Yam exosomes can significantly improve the symptoms of DSS-induced colitis. The experimental results showed that the disease activity index (DAI) of the intervention group was significantly lower than that of the model group, and the expression levels of pro-inflammatory factors TNF-α, IL-1β and IL-6 in colon tissue were significantly inhibited, which confirmed its excellent anti-inflammatory activity.

[0019] (2) Yam exosomes can not only prevent colitis, but also effectively improve the symptoms of constipation induced by loperamide. The mechanism is to bidirectionally regulate the expression of gastrointestinal hormones: by reducing the expression of intestinal inhibitory peptide (VIP) and endothelin-1 (ET-1), which have the function of inhibiting intestinal peristalsis, while upregulating the expression of motilin (MTL) and substance P (SP), which promote intestinal motility, the intestinal excretion function is improved at the molecular level.

[0020] (3) As a plant that is both food and medicine, yam is rich in raw materials and has high safety. Its extracted exosomes, as a natural nanovesicle, not only have good biocompatibility, but also have the potential advantage of low toxicity and side effects, making them suitable for long-term intervention and preventive application.

[0021] (4) The present invention uses polyethylene glycol (PEG) precipitation combined with ultracentrifugation to extract yam exosomes. Compared with the traditional sucrose density gradient centrifugation method, the operation process is simpler and can efficiently obtain yam exosomes with uniform morphology and complete structure. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a transmission electron microscope image of yam exosomes.

[0023] Figure 2 This is a particle size analysis diagram of yam exosomes.

[0024] Figure 3Figure 1 shows the results of a study on the mechanism by which yam exosomes alleviate DSS-induced colitis by regulating inflammation levels.

[0025] Figure 4 This image shows the effect of yam exosomes on improving intestinal excretion function in a loperamide-induced constipation model.

[0026] Figure 5 Figure showing the results of a study on the molecular mechanism by which yam exosomes alleviate loperamide-induced constipation.

[0027] Figure 6 To investigate the effects of different interventions on the DAI index and TNF-α in a DSS-induced colitis model.

[0028] Figure 7 To investigate the effects of different interventions on the DAI index in a DSS-induced colitis model.

[0029] Figure 8 To investigate the effects of different interventions on the expression of the inflammatory factor TNF-α in a DSS-induced colitis model.

[0030] Figure 9 To investigate the effects of different interventions on the expression of the inflammatory cytokine IL-1β in a DSS-induced colitis model.

[0031] Figure 10 To investigate the effects of different interventions on the expression level of the inflammatory cytokine IL-6 in a DSS-induced colitis model. Detailed Implementation

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0033] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0035] The raw materials used in this invention are: fresh yam (commercially available), polyethylene glycol 6000 (PEG 6000, analytical grade, purchased from Beijing Solarbio Science & Technology Co., Ltd.), sodium chloride (NaCl, food grade, commercially available), phosphate buffer (PBS, pH 7.2-7.4, Wuhan Sewell Biotechnology Co., Ltd.), and BCA protein concentration assay kit (Suzhou Xinsaimei Biotechnology Co., Ltd.).

[0036] The instruments used in this invention embodiment are: high-speed refrigerated centrifuge (OHAUS, FC5718R), ultracentrifuge (Beckman Coulter, Optima-XPN-100), and Beckman 14mL centrifuge tubes.

[0037] The method for detecting the disease activity index (DAI) in this embodiment of the invention: DAI (Defecal Incidence Artery Disease) is assessed in mice based on clinical symptoms (weight loss rate, fecal morphology, and fecal occult blood) (specific scoring criteria are shown in Table 1). During the modeling period, mouse weight was measured daily, and fecal blood loss and morphology were tested. Scoring was performed according to Table 1. DAI is the sum of the three scores: DAI = weight loss score + fecal morphology score + fecal occult blood score. Fecal occult blood was detected using a fecal occult blood qualitative test kit. The specific procedure was performed according to the kit instructions. If reddish-brown or bright red blood was visible in the feces, it was considered gross hematochezia. Fecal morphology was classified into five grades: normal, soft stool, loose stool, mucus-like stool, watery stool, and diarrhea.

[0038] Table 1 Disease Activity Index Scoring Criteria

[0039] RNA extraction and real-time quantitative PCR in this embodiment of the invention: RNA was extracted from colon tissue (50 mg) using TRIzol reagent (Beijing Qingke Biotechnology Co., Ltd., China). RNA concentration was then determined using ultra-micro spectrophotometry, and purity was evaluated by the A260 / A280 ratio. Total RNA (2 μg) was reverse transcribed into cDNA using the MightyScript First-Strand cDNA Synthesis Master Mix Kit (Sangon Biotech). For tissue samples from the DSS model group, isolated RNA was purified using lithium chloride (LiCI). Subsequently, 0.1 volume of 3 M sodium acetate (Beyotime) and 2 volumes of ethanol were added to 1 volume of RNA solution to reprecipitate RNA in the absence of DSS. Real-time quantitative PCR (qPCR) was performed according to the manufacturer's protocol using a Roche LightCycler 480 II (Indianapolis, IN, USA).

[0040] Example 1 This embodiment provides a method for extracting and purifying yam exosomes, including the following steps: (1) Wash the fresh yam raw material, peel it, chop it, mix it with PBS buffer at 4℃ and blend it into a paste, filter it and take the filtrate; the mass ratio of PBS buffer to yam is 2:1. (2) Centrifuge the filtrate obtained in step 1 three times at 4°C. The centrifugation conditions are 2000 rpm, 10 min, 6000 rpm, 30 min, 10000 rpm, and 40 min respectively. Discard the precipitate and collect the supernatant. (3) Take the supernatant from step 2 and centrifuge it at 75000g for 1h at 4℃ to collect the supernatant; (3) Mix the supernatant obtained in step 3 with 24% PEG6000 at a 1:1 ratio and let it stand at 4°C for 24 hours.

[0041] (4) After standing, the mixture obtained in step 4 is centrifuged at 4°C, 10,000 rpm for 1 hour, and the precipitate is taken. The precipitate is yam exosomes. It is resuspended with PBS buffer to form a suspension.

[0042] Take 20 μL of the exosomes obtained above and drop them onto a copper grid for adsorption for 5 min, then absorb the floating liquid with filter paper; take 20 μL of phosphotungstic acid and drop it onto a copper grid for adsorption for 3 min, then absorb the floating liquid with filter paper; dry at room temperature for several minutes; perform electron microscopy at 100 kV to obtain transmission electron microscopy imaging results, such as... Figure 1 As shown. Figure 1 This is a transmission electron microscope image of yam exosomes taken at a magnification of 40,000.

[0043] Take 10 μL of yam exosomes and dilute them to an appropriate concentration. After the instrument performance test using a standard is passed, the exosome sample can be loaded. Note that serial dilution is necessary to avoid clogging the injection needle.

[0044] Once the sample is tested, the particle size and concentration information of the exosomes detected by NTA can be obtained, such as... Figure 2 As shown, the average particle size of yam exosomes is 80 nm.

[0045] Example 2 This embodiment studies the mechanism by which DSS-induced colitis is alleviated by regulating inflammation levels, specifically as follows: NC: Normal group; M: Colitis model group; each mouse was given 2% DSS drinking water for 4 days, then normal DSS-free drinking water for 7 days, and this cycle was repeated for four cycles, with no recovery period in the last cycle.

[0046] YEN-L: Low-dose intervention group of yam exosomes; during modeling, each mouse was administered 30 mg / kg / d of hawthorn exosomes by gavage.

[0047] YEN-H: High-dose intervention group of yam exosomes; during modeling, each mouse was administered 60 mg / kg / d of hawthorn exosomes by gavage.

[0048] During the experiment, mouse weight was recorded at the end of each intervention and recovery period and within 16 days before the end of the experiment. Mouse feces were collected and fecal occult blood tests were performed.

[0049] After fasting for 12 hours but not water, the mice were euthanized. The colons of the euthanized mice were placed on filter paper, and their length was measured and photographed.

[0050] like Figure 3 As shown, (a) represents the weight changes of mice in different groups; (b) represents the colon length of mice in different groups; (c) represents the disease activity index of mice in different groups; (d) represents the fecal morphology of mice in different groups; (e) represents the expression level of the inflammatory cytokine TNF-α in the colon tissue of mice in different groups; (f) represents the expression level of the inflammatory cytokine IL-1β in the colon tissue of mice in different groups; (g) represents the expression level of the inflammatory cytokine IL-6 in the colon tissue of mice in different groups; (h) represents the expression level of ZO-1 in the colon tissue of mice in different groups; (i) represents the expression level of MUC-1 in the colon tissue of mice in different groups; and (j) represents the expression level of MUC-2 in the colon tissue of mice in different groups. ("#" indicates that compared with the NC group, # indicates p<0.05, ## indicates p<0.01; " "This indicates that compared to group M, HEN-L and HEN-H" This indicates that p < 0.05. (Indicates p < 0.01) Depend on Figure 3 It can be seen that, compared with the normal group (NC), the body weight of mice in the colitis model group (M group) was significantly reduced. Figure 3 a) The Disease Activity Index (DAI) increased significantly ( Figure 3 b) The length of the colon is significantly shortened ( Figure 3 c) Deterioration of stool morphology accompanied by obvious rectal bleeding ( Figure 3 d), and at the same time, the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 in colon tissue all showed a significant abnormal increase ( Figure 3 e.g., at the same time, the expression of tight junction protein ZO-1 and mucins MUC-1 and MUC-2 was significantly decreased. Figure 3 hj); After intervention with different doses of yam exosomes (YEN-L and YEN-H groups), the trend of weight loss in mice was significantly alleviated, the DAI score was significantly reduced, colon length was significantly restored, fecal characteristics were significantly improved, and the abnormal expression levels of TNF-α, IL-1β, IL-6, and IL-10 in colon tissue were reversed, while the expression of ZO-1, MUC-1, and MUC-2 was significantly upregulated. Figure 3 hj).

[0051] This indicates that yam exosomes can significantly improve DSS-induced colitis symptoms in mice by inhibiting the expression of inflammatory factors and repairing the intestinal barrier and mucus layer.

[0052] Example 3 This embodiment explores how yam exosomes alleviate loperamide-induced constipation by regulating gastrointestinal hormones. Specifically: NC: Normal group, administered an equal volume of phosphate buffer solution by gavage daily; M: Constipation model group; each mouse was administered loperamide by gavage at a dose of 10.0 mg / kg / d.

[0053] YEN-L: Low-dose intervention group of yam exosomes; each mouse was administered loperamide at gavage and yam exosomes at a dose of 10.0 mg / kg / d and 30 mg / kg / d respectively.

[0054] YEN-H: High-dose intervention group of yam exosomes; each mouse was administered loperamide at gavage and yam exosomes at a dose of 10.0 mg / kg / d and 60 mg / kg / d respectively.

[0055] Each group received intervention for five weeks.

[0056] like Figure 4As shown, (a) is the fecal weight within 6 hours, (b) is the fecal water content, (c) is the time to the first blue stool, (d) is the small intestinal propulsion rate, and (e) is the fecal particle size within 6 hours. ("#" indicates comparison between group M and group NC; # indicates p < 0.05, ## indicates p < 0.01; " "This indicates that compared to group M, groups YEN-L and YEN-H..." This indicates that p < 0.05. This indicates that p < 0.01.

[0057] Depend on Figure 4 It can be seen that, compared with the normal group (NC), the fecal weight of mice in the constipation model group (M group) within 6 hours was significantly lower. Figure 4 a) Fecal moisture content ( Figure 4 b) Small intestinal propulsion rate ( Figure 4 d) and the number of stool particles excreted within 6 hours ( Figure 4 e) all showed a significant decrease, and the time to excretion of the first blue stool was significantly prolonged ( Figure 4 c) indicates that the loperamide-induced mouse constipation model was successfully established, and the mice showed severe impairment of intestinal excretion and transit functions; After intervention with yam exosomes (YEN-L and YEN-H groups), the fecal weight, fecal water content, small intestinal propulsion rate, and number of fecal particles in the model mice were significantly increased, and the time to excretion of the first blue feces was greatly shortened. These results directly demonstrate from the phenotypic and intestinal function levels that yam exosomes can significantly improve the intestinal excretion function of constipated mice and effectively increase the intestinal transit rate.

[0058] like Figure 5 As shown, (a) is the expression level of intestinal inhibitory peptide in colon tissue, (b) is the expression level of intestinal inhibitory peptide in serum, (c) is the expression level of motilin in colon tissue, (d) is the expression level of endothelin-1 in colon tissue, (e) is the expression level of endothelin-1 in serum, (f) is the expression level of motilin in serum, (g) is the expression level of substance P in colon tissue, and (h) is the expression level of substance P in serum.

[0059] Depend on Figure 5 It can be seen that, compared with the normal group (NC), the colon tissue and serum of the constipation model group (M group) mice contained higher levels of intestinal inhibitory peptide (VIP). Figure 5 a, 5b) and endothelin-1 (ET-1, Figure 5 The expression levels of d and 5e were significantly and abnormally elevated, while the expression levels of motilin (MTL) in colon tissue and serum were significantly elevated. Figure 5 c, 5f) and P substance (SP, Figure 5 The expression levels of g and 5h were significantly reduced, indicating that there was a significant gastrointestinal hormone secretion disorder in the constipation model mice. After intervention with yam exosomes (YEN-L and YEN-H groups), the abnormally elevated levels of intestinal inhibitory peptide (VIP) and endothelin-1 (ET-1) in mice were effectively inhibited and downregulated, while the abnormally reduced levels of motilin (MTL) and substance P (SP) were effectively restored and increased.

[0060] This result clarifies at the molecular level that yam exosomes can effectively relieve constipation symptoms by bidirectionally regulating the expression of gastrointestinal hormones (i.e., inhibiting VIP and ET-1, which have a slowing effect on peristalsis, while promoting MTL and SP, which increase intestinal motility).

[0061] In conclusion, Figure 4 Functional phenotypic factors, such as stool volume, stool characteristics, and intestinal transit rate, confirmed that yam exosomes can improve intestinal excretion function in constipated mice. Figure 5 Revealing its essential mechanism of action at the molecular level, by regulating gastrointestinal hormones to balance intestinal motility and secretory function, and ultimately relieving constipation, both studies clarify the effectiveness and mechanism of yam exosome intervention in constipation.

[0062] Comparative Example 1 The difference between this embodiment and Embodiment 2 is that the yam exosomes are replaced with yam water extract, while the rest of the steps are the same as in Embodiment 2.

[0063] The difference between this comparative example and Example 2 is that the yam exosomes are replaced with an equal dose of yam water extract (yam polysaccharide, YP), while the other steps are the same as in Example 2.

[0064] like Figure 6 As shown, (a) shows the effect of yam exosome intervention on the disease activity index (DAI) of DSS-induced colitis model mice; (b) shows the effect of yam polysaccharide (YP) intervention on the disease activity index (DAI) of DSS-induced colitis model mice; (c) shows the effect of yam exosome intervention on the expression level of TNF-α in colon tissue; and (d) shows the effect of yam polysaccharide (YP) intervention on the expression level of TNF-α in colon tissue.

[0065] The results showed that, in the DSS-induced colitis model, yam-derived exosomes (YEN) demonstrated significantly superior potential compared to homologous yam aqueous extract (YP) in improving the disease activity index (DAI) and remodeling the immune microenvironment.

[0066] The data comparison in Figures (a) and (b) shows that the DAI index of the model group (M / DSS) increased significantly after modeling. After intervention, the DAI index of each dose group of yam exosomes could be effectively controlled at around 2.0, while the DAI score of the yam polysaccharide group (DSS+YP) remained at around 2.5 after intervention, indicating that yam exosomes have a stronger effect on alleviating the overall condition of colitis.

[0067] Regarding the inhibition of key pro-inflammatory factors, the data in Figures (c) and (d) show that the yam exosome intervention group (YEN) was able to inhibit the expression of TNF-α in colon tissue to a greater extent, restoring its relative expression level to near the level of the normal control group (NC). In contrast, the yam polysaccharide group showed a significantly weaker reduction in TNF-α absorbance and control over the inflammatory response compared to the exosome group.

[0068] In conclusion, yam exosomes are significantly more effective than traditional yam water extracts in relieving colitis symptoms and downregulating inflammatory markers. This is mainly due to the fact that yam exosomes, as natural nanovesicle structures, have better biocompatibility and targeted delivery advantages, and can more accurately protect and deliver active ingredients to the intestinal lesion area.

[0069] Comparative Example 2 The difference between this comparative example and Example 2 is that the yam exosomes were replaced with extracellular vesicles derived from galangal (KGEVs), ginger exosomes (GELNs), nanobiota derived from Sichuan pepper (ZbELNs), and Sophora flavescens-derived exosome nanovesicles loaded with CX5461 (SFELNVs@CX5461).

[0070] like Figure 7 As shown, (a) shows the effect of yam exosomes (YEN) intervention on the disease activity index (DAI) of DSS-induced colitis model mice; (b) shows the intervention effect of kaempferia-derived extracellular vesicles (KGEVs); (c) shows the intervention effect of ginger-derived exosomes (GELNs); (d) shows the intervention effect of zanthoxylum bungeanum-derived nanobiological agents (ZbELNs); and (e) shows the intervention effect of Sophora flavescens-derived exosome nanovesicles loaded with CX5461 (SFELNVs@CX5461).

[0071] The results showed that, in the DSS-induced colitis model, yam-derived exosomes (YEN) demonstrated superior therapeutic effects in improving the disease activity index (DAI) compared to the other four plant-derived exosomes.

[0072] Regarding the disease activity index, the high-dose YEN group ( Figure 7a) It exhibits a very strong ability to reverse symptoms, significantly reducing the peak value of the disease activity index to around 2.0; while in the comparative data, the ZbELNs source of Sichuan pepper ( Figure 7 d) Even after the intervention ended, the DAI score remained at a level close to 8.0, indicating a severe disease state. GELNs derived from ginger ( Figure 7 c) also maintained a rating above 4.0, with kaempferia source KGEVs ( Figure 7 (b) Although showing a downward trend, the final score remained close to 5.0. Even compared to matrine-derived vesicles loaded with synthetic drugs ( Figure 7 e) In comparison, the natural yam exosomes of this invention also have a significant competitive advantage in terms of the depth of clinical symptom relief.

[0073] like Figure 8 As shown, (a) shows the effect of yam exosomes (YEN) on TNF-α expression in the DSS-induced colitis model; (b) shows the effect of kaempferia-derived extracellular vesicles (KGEVs) on TNF-α expression; (c) shows the effect of zanthoxylum bungeanum-derived nanobioreactors (ZbELNs) on TNF-α expression; and (d) shows the effect of Sophora flavescens-derived exosome nanovesicles loaded with CX5461 (SFELNVs@CX5461) on TNF-α expression.

[0074] like Figure 9 As shown, (a) the effect of yam exosomes (YEN) on IL-1β expression; (b) the effect of kaempferia-derived extracellular vesicles (KGEVs) on IL-1β expression; (c) the effect of ginger-derived exosomes (GELNs) on IL-1β expression; (d) the effect of Sophora flavescens-derived exosome nanovesicles loaded with CX5461 (SFELNVs@CX5461) on IL-1β expression; and (e) the effect of Zanthoxylum bungeanum-derived nanobioreactors (ZbELNs) on IL-1β expression.

[0075] like Figure 10 As shown, (a) shows the effect of yam exosomes (YEN) on IL-6 expression; (b) shows the effect of kaempferia-derived extracellular vesicles (KGEVs) on IL-6 expression; (c) shows the effect of ginger-derived exosomes (GELNs) on IL-6 expression; (d) shows the effect of Sichuan pepper-derived nanobioreactors (ZbELNs) on IL-6 expression; and (e) shows the effect of Sophora flavescens-derived exosome nanovesicles loaded with CX5461 (SFELNVs@CX5461) on IL-6 expression.

[0076] The results showed that yam exosomes exhibited a more precise downregulation effect on pro-inflammatory factors (TNF-α, IL-1β, IL-6). Data comparison revealed that the YEN group significantly reduced the expression levels of pathologically elevated pro-inflammatory factors in colon tissue or serum, bringing them close to the levels of the normal control group (NC). While kaempferia-derived KGEVs and Sichuan pepper-derived ZbELNs inhibited inflammatory factors, their post-intervention expression levels remained significantly higher than the normal baseline. Particularly in inhibiting the pro-inflammatory factor IL-1β, yam exosomes were significantly more effective than ginger-derived GELNs and Sichuan pepper-derived ZbELNs in restoring the expression level to baseline.

[0077] In summary, the yam exosomes of this invention outperform the aforementioned plant-derived exosomes in terms of their actual quantitative performance in three core biological dimensions: reducing the DAI index, blocking the pro-inflammatory cascade, and inducing endogenous anti-inflammatory regulation. This demonstrates that yam exosomes extracted using the specific process of this invention possess unexpectedly high biological activity and represent a significant technological advancement.

[0078] Comparative Example 3 This comparative example uses traditional ultracentrifugation to extract yam exosomes, aiming to evaluate the technical effectiveness of this invention in processing the special matrix of yam by comparing it with the method of this invention. The specific steps are as follows: Fresh yam raw materials were washed, peeled, and chopped. Pre-cooled PBS buffer was added at a mass ratio of 1:2, and the mixture was mechanically juiced and filtered to obtain crude yam juice. The filtrate was centrifuged at 1000×g for 10 min and 3000×g for 15 min at 4°C to remove coarse impurities, followed by centrifugation at 10000×g for 30 min to remove cell debris and some starch granules. The supernatant was collected. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 120000×g for 2 h. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS buffer and washed again by centrifugation at 120000×g for 1 h. Finally, the precipitate was resuspended in PBS to obtain yam exosome samples.

[0079] Under the same starting material conditions, the performance of the products obtained in this comparative example and Example 1 of the present invention was tested (the number of particles measured by nanoparticle tracking analysis was used as the evaluation index). The results are shown in Table 2. The PEG precipitation method described in the present invention is significantly better than that of comparative example 3 in terms of exosome extraction efficiency, and its unit yield is increased by about 30 times.

[0080] Table 2

[0081] Analysis suggests that this difference may be related to the compositional characteristics of the yam matrix. Yam tubers are rich in mucoproteins and amylopectin, which readily form a viscoelastic network structure in aqueous systems, thereby encapsulating exosome nanovesicles. During the ultracentrifugation process described in Comparative Example 3, relying solely on centrifugal force for separation is insufficient to completely overcome the matrix's encapsulation effect on exosomes, resulting in some exosomes failing to settle effectively or being lost along with impurities, thus affecting the recovery rate.

[0082] In contrast, the method of the present invention introduces polyethylene glycol (PEG 6000) and utilizes its size exclusion effect to change the microenvironment of the system, which helps to weaken the interaction between exosomes and the matrix, promotes the release of exosomes from the complex system and their aggregation and sedimentation, thereby improving the extraction efficiency.

[0083] Furthermore, from the perspective of product quality and process applicability, the long-term high-speed ultracentrifugation conditions used in Comparative Example 3 may have a certain impact on the exosome membrane structure; while the method of the present invention has relatively mild conditions, which is conducive to maintaining the integrity of the exosome structure and its biological activity.

[0084] Meanwhile, Comparative Example 3 relies heavily on ultracentrifugation equipment, which has problems such as limited processing capacity and high energy consumption, making it unsuitable for large-scale application; while the method of the present invention has a relatively simple operation process and can be implemented with conventional equipment, which helps to reduce production costs and improve industrial feasibility.

[0085] In summary, the method described in this invention demonstrates superior performance in terms of yam exosome extraction efficiency, structure preservation, and process applicability.

[0086] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. The application of yam exosomes in the preparation of drugs for the prevention of colitis, characterized in that: The drug contains an effective dose of yam exosomes; The yam exosomes have at least one of the following effects: a. Significantly repairs damaged intestinal mucosal barrier; b. Reduces the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in colon tissue; c. Regulate the expression of gastrointestinal hormones, including reducing VIP and ET-1 expression and increasing MTL and SP expression. d. Improves intestinal excretion function, including increasing stool weight and water content, shortening the time to the first blue stool, improving small intestinal propulsion rate, and increasing the number of stool particles within 6 hours.

2. Use according to claim 1, characterized in that: The mass percentage of yam exosomes in the drug is 2% to 15%.

3. Use according to claim 1, characterized in that: The dosage form of the drug is retention enema solution, enteric microcapsule, nanogel or lyophilized powder.

4. The application as described in claim 1, characterized in that: The method for extracting yam exosomes includes the following steps: Wash, peel, and chop the fresh yam raw material. Mix it with PBS buffer at 4℃ and blend it into a paste. Filter the paste and collect the filtrate. Centrifuge the obtained filtrate at differential speed, discard the precipitate, and collect the supernatant; Take the supernatant and centrifuge it at ultraspeed to collect the supernatant; The supernatant after ultracentrifugation was mixed with 24% PEG6000 and allowed to stand at 4°C for 24 hours. After standing, the resulting mixture was centrifuged at 4℃, 10000rpm for 1h, and the precipitate was collected. The precipitate was yam exosomes.

5. The application as described in claim 4, characterized in that: The differential centrifugation was performed under the following conditions: three centrifugations at 4°C, with the centrifugation conditions being 2000 rpm for 10 min, 6000 rpm for 30 min, and 10000 rpm for 40 min in sequence.

6. The application as described in claim 4, characterized in that: The mass ratio of the PBS buffer to the yam is 2:

1.

7. The application as described in claim 4, characterized in that: The volume ratio of the supernatant after ultracentrifugation to PEG6000 is 1:

1.

8. The application as described in claim 1, characterized in that: The colitis referred to is ulcerative colitis, radiation enteritis, or intestinal mucosal damage induced by chemotherapy drugs.

9. A pharmaceutical composition for the prevention or treatment of colitis, characterized in that: It includes yam exosomes prepared by any one of claims 4 to 7, and pharmaceutically acceptable carriers or excipients.

10. A pharmaceutical composition for repairing the intestinal mucosal barrier, characterized in that: It includes yam exosomes prepared by any one of claims 4 to 7, and pharmaceutically acceptable carriers or excipients.

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