Astragalus extracellular vesicles and vesicle lipid particles with anti-colitis effects, and methods of making and uses thereof

Abstract

The present application belongs to the field of medicine, and particularly relates to astragalus membranaceus extracellular vesicles and vesicle lipid particles with an anti-colitis effect and a preparation method and use thereof. The present application first discovers that the astragalus membranaceus extracellular vesicles and vesicle lipid particles have a significant prevention and treatment effect on DSS-induced colitis, and are safe to use and do not cause adverse reactions, so the astragalus membranaceus extracellular vesicles and vesicle lipid particles are ideal candidates for developing drugs for preventing or treating colitis, and have high commercial value and social significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to astragalus extracellular vesicles and vesicle lipid particles with anti-colitis effects and a preparation method and uses thereof. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a chronic, non-specific, inflammatory gastrointestinal disease, mainly including Crohn's disease (CD) and ulcerative colitis (UC). The main symptoms of Crohn's disease are chronic diarrhea and abdominal pain, while patients with ulcerative colitis are more likely to have intermittent abdominal cramps and bloody diarrhea. According to the statistics of the China Center for Disease Control and Prevention, the total number of IBD cases in China is about 350,000, and it is estimated that by 2025, the total number of patients will exceed 1.5 million. The etiology and pathogenesis of IBD are not clear, and most scholars believe that it may be related to genetic susceptibility, environmental changes, immune regulation, and microecological disorders.

[0003] The current treatment methods for IBD mainly include drugs, surgery, nutritional support, etc. Drugs mainly include tumor necrosis factor inhibitors, aminosalicylates, immunomodulators, and corticosteroids. 5-amino salicylic acid and glucocorticoids, immunosuppressants, etc. are commonly used in clinical treatment of UC, but have low cure rate, easy recurrence, etc. and long-term use can produce some adverse reactions, such as intolerance or allergic reactions. Mesalazine, also known as 5-amino salicylic acid, is the active ingredient of sulfasalazine (SASP) for the treatment of UC. Mesalazine can inhibit the synthesis of prostanoids and the formation of inflammatory mediators leukotrienes, thereby significantly inhibiting inflammation of the intestinal mucosa, and can be used for ulcerative colitis, ulcerative rectitis, and Crohn's disease. However, mesalazine also has corresponding defects, and the more common ones are some gastrointestinal and extra-intestinal effects, such as nausea, abdominal pain, diarrhea, and headache. There is also a possibility of serious side effects, including bone marrow toxicity with leukopenia and agranulocytosis, thrombocytopenia, aplastic anemia, extra-intestinal inflammation, and nervous system manifestations, the most worrying but rare problem is renal toxicity caused by interstitial nephritis.

[0004] China has abundant resources of animal and plant Chinese herbal medicines. Due to the characteristics of "multi-component, multi-target, multi-pathway and overall regulation", Chinese herbal medicines have become one of the research hotspots of anti-UC drugs in recent years. Studies have shown that Chinese herbal medicines, including active ingredients, single Chinese herbal medicines and their extracts, and traditional Chinese medicine compounds, can effectively improve the symptoms of UC; and regulating intestinal flora imbalance is an important potential target for treating UC. For example, berberine can increase lactic acid-producing bacteria and carbohydrate-hydrolyzing bacteria, and reduce conditional pathogenic bacteria; phellopterin can regulate the composition of intestinal flora; Periplaneta americana extract and Huanglian Jiedu Decoction can increase beneficial bacteria such as Lactobacillus and reduce colon inflammation. In traditional Chinese medicine, it is generally believed that spleen and stomach qi deficiency is the root cause of ulcerative colitis, so the basic treatment method in clinical treatment is to invigorate the spleen and replenish qi. Studies have shown that total polysaccharides from Schisandra chinensis can significantly improve the inflammatory damage of the colon, reduce the spleen index, increase the thymus index, and effectively reduce the content (activity) of MPO, NO, MDA and ROS in the colon, while increasing the content of GSH and SOD. In addition, Schisandra chinensis polysaccharides regulate the composition and diversity of intestinal microorganisms in colitis mice, significantly increase the relative abundance of Firmicutes, Glycobacterium, Enterorhabdus, etc., reduce the relative abundance of intestinal bacteria such as Firmicutes, Turicibacter and Akkermansia, and significantly increase the content of propionic acid, butyric acid and isobutyric acid in the colon, suggesting that total polysaccharides from Schisandra chinensis can be used to prepare drugs for treating or improving inflammatory bowel disease. Schisantherin A is a bioactive lignan isolated from the fruit of Schisandra chinensis. Studies have shown that Schisantherin A has anti-inflammatory, anti-free radical, liver-protecting and inhibition of gastrointestinal smooth muscle spasm effects. Studies have shown that Schisantherin A has a protective effect on trinitrobenzene sulfonic acid-induced ulcerative colitis in mice and experimental colitis in rats.

[0005] In recent years, researchers have gradually discovered and isolated plant exosomes and extracellular vesicles with a particle size of 30-150 nm from plants and characterized them. Data shows that plant exosomes and extracellular vesicles may play an important role in maintaining intestinal symbiosis and internal environment stability. However, extracellular vesicles from different plant sources have great differences, and their biological functions are also different. The efficacy of the original plant and its extracts or natural product monomers cannot generally be expected to predict the effect of plant-derived extracellular vesicles, so it is difficult to find safe and effective plant-derived extracellular vesicles for treating colitis. SUMMARY

[0006] In view of the above situation, in order to solve the defects of the prior art, the present application provides astragalus extracellular vesicles and vesicle lipid particles with anti-colitis effect and a preparation method and use thereof.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a pharmaceutical composition having a preventive or therapeutic effect on colitis, the pharmaceutical composition comprising Astragalus membranaceus extracellular vesicles.

[0009] Preferably, the Astragalus membranaceus extracellular vesicles are nanoscale vesicles.

[0010] In a second aspect, the present application provides a pharmaceutical composition having a preventive or therapeutic effect on colitis, the pharmaceutical composition comprising Astragalus membranaceus extracellular vesicle lipid particles.

[0011] Preferably, the Astragalus membranaceus extracellular vesicle lipid particles are nanoscale.

[0012] As an optional mode, in the above pharmaceutical composition, the Astragalus membranaceus extracellular vesicles include one or more of the following: exosomes, apoptotic bodies, microvesicles, lysosomes, endosomes, lipoproteins, or cell-derived nanoparticles.

[0013] Preferably, the Astragalus membranaceus extracellular vesicles have a particle size of 50-300 nm.

[0014] As an optional mode, in the above pharmaceutical composition, the Astragalus membranaceus extracellular vesicles are double-membrane structures under transmission electron microscopy, are mostly concave round cake-shaped, and have a particle size of 50-150 nm.

[0015] As an optional mode, in the above pharmaceutical composition, the Astragalus membranaceus extracellular vesicles are prepared using a PEG sedimentation method, which includes the following steps: Astragalus membranaceus is preliminarily washed with tap water at room temperature, then washed with PBS solution for 3 times, the washed Astragalus membranaceus is cut into a sheet shape with a thickness of about 5 mm, soaked with an appropriate amount of PBS, crushed in a wall breaking machine, filtered through a gauze, remove the Astragalus membranaceus residue, collect the Astragalus membranaceus juice, under the condition of 4℃, the filtered Astragalus membranaceus juice is sequentially centrifuged at 3,000g for 30 min to collect the supernatant, then the collected supernatant is centrifuged at 5,000g for 40 min to collect the supernatant, then the collected supernatant is centrifuged at 15,000g for 30 min, then the supernatant is sedimented overnight using 8% PEG6000 containing 500mM NaCl, the sedimented Astragalus membranaceus supernatant is centrifuged at 5,000g for 30 min to obtain a precipitate, the precipitate is resuspended with a filtered sterilized PBS solution, the pH is adjusted to neutral, and then the Astragalus membranaceus extracellular vesicles are obtained.

[0016] As an optional mode, in the above-mentioned pharmaceutical composition, the Astragalus membranaceus extracellular vesicles are prepared by differential-ultracentrifugation combined with sucrose density gradient centrifugation, to obtain more pure Astragalus membranaceus extracellular vesicles, which comprises the following steps: the Astragalus membranaceus is preliminarily washed with tap water at room temperature, then washed with PBS solution for 3 times, the washed Astragalus membranaceus is cut into pieces with a thickness of about 5 mm, and then soaked in appropriate amount of PBS solution, after sufficient soaking, the Astragalus membranaceus is broken in a wall-breaking machine, and then filtered through a gauze to remove the Astragalus membranaceus residues, and the Astragalus membranaceus juice is collected, and then the filtered Astragalus membranaceus juice is sequentially centrifuged at 3,000g for 30 min to collect the supernatant, then the collected supernatant is centrifuged at 5,000g for 40 min to collect the supernatant, then the collected supernatant is centrifuged at 15,000g for 30 min to collect the supernatant, then the collected supernatant is centrifuged at 150,000g for 70 min to obtain a precipitate, the precipitate is resuspended with PBS solution filtered by sterilization, and then Astragalus membranaceus extracellular vesicles are preliminarily obtained, and then relatively pure Astragalus membranaceus extracellular vesicles are obtained by sucrose density gradient centrifugation at 20,000g for 2h.

[0017] As an optional mode, in the above-mentioned pharmaceutical composition, the preparation method of the Astragalus membranaceus extracellular vesicle lipid particles comprises the following steps: a mixed solution of methanol and chloroform is taken in a clean glass test tube, an Astragalus membranaceus extracellular vesicle suspension is added, and then vortexed for 1 min, a mixed solution of chloroform and pure water is added, and then vortexed for 1 min, then centrifuged, the lower organic phase is taken out by a glass pipette and transferred to a new glass test tube, dried by nitrogen blowing, PBS solution is added to the dried glass test tube, ultrasonically treated in a water bath for 5 min, filtered through a 0.45μm filter, and then Astragalus membranaceus extracellular vesicle lipid particles are obtained.

[0018] More preferably, the preparation method of the Astragalus membranaceus extracellular vesicle lipid particles comprises the following steps: 4mL of methanol and 2mL of chloroform are taken in a clean glass test tube, 1mL (containing 5x10 10 -1x10 12 6 of Astragalus membranaceus extracellular vesicle suspension is added, and then vortexed for 1 min, 2mL of chloroform and 2mL of pure water are added, and then vortexed for 1 min, then centrifuged at 2300rpm for 10 min, the lower organic phase is taken out by a glass pipette and transferred to a new glass test tube, dried by nitrogen blowing, 600μL of PBS solution is added to the dried glass test tube, ultrasonically treated in a water bath for 5 min, filtered through a 0.45μm filter, and then Astragalus membranaceus extracellular vesicle lipid particles are obtained.

[0019] As an optional mode, in the above pharmaceutical composition, the pharmaceutical composition further comprises other clinically commonly used drugs for preventing or treating colitis, and the other clinically commonly used drugs for preventing or treating inflammatory bowel disease are selected from one or more of the following: antibiotics, 5-aminosalicylic acid (5-ASA) (such as sulfasalazine or mesalazine), corticosteroids (such as prednisone), immunomodulators (such as azathioprine or methotrexate), or biological products (such as infliximab, adalimumab, certolizumab pegol, or natalizumab).

[0020] In a third aspect, the present application provides a pharmaceutical preparation having a preventive or therapeutic effect on colitis, the pharmaceutical preparation comprising the pharmaceutical composition of the first aspect or the second aspect described above, and a pharmaceutically acceptable carrier.

[0021] As an optional mode, in the above pharmaceutical preparation, the pharmaceutical preparation is an oral preparation, an injection, or a rectal administration preparation.

[0022] Preferably, the oral preparation is selected from an oral liquid, a tablet, a powder, a capsule, or a granule.

[0023] In a fourth aspect, the present application provides use of the pharmaceutical composition of the first aspect or the second aspect described above or the pharmaceutical preparation of the third aspect described above in the preparation of a drug for preventing or treating colitis.

[0024] As an optional mode, in the above use, the colitis is ulcerative colitis or Crohn's disease.

[0025] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described below (such as the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be repeated one by one here.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] (1) The present application first discovers that Astragalus membranaceus extracellular vesicles and vesicle lipid particles have a significant preventive and therapeutic effect on DSS-induced colitis, and are safe to use and do not produce adverse reactions, so that Astragalus membranaceus extracellular vesicles and vesicle lipid particles are ideal candidates for developing drugs for preventing or treating colitis, and have high commercial value and social significance.

[0028] (2) The Astragalus membranaceus extracellular vesicles and vesicle lipid particles of the present application are easy to obtain, have a simple extraction method, and are low in cost. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1TEM structure characterization of Astragalus membranaceus extracellular vesicles prepared in Example 1. Wherein, the scale in the figure is 100 nm.

[0030] Figure 2 Particle size distribution of several traditional Chinese medicine extracellular vesicles prepared in Example 1.

[0031] Figure 3 Zeta potential distribution of Astragalus membranaceus extracellular vesicles prepared in Example 1.

[0032] Figure 4 Disease activity index (DAI) changes of mice in each group in Example 1.

[0033] Figure 5 Colon length changes of mice in each group in Example 1.

[0034] Figure 6 Organ coefficients of mice in each group. Wherein, Figure 6 A is the spleen coefficient, Figure 6 B is the kidney coefficient.

[0035] Figure 7 Schematic diagram of differential centrifugation combined with sucrose density to obtain Astragalus membranaceus extracellular vesicles.

[0036] Figure 8 TEM structure characterization of Astragalus membranaceus extracellular vesicles prepared in Example 2. Wherein, the left figure is the morphology of multiple extracellular vesicles gathered under low magnification, and the right figure is the extracellular vesicle with clear membrane structure under high magnification, and the scale in the figure is 100 nm.

[0037] Figure 9 Particle size distribution of Astragalus membranaceus extracellular vesicles prepared in Example 2.

[0038] Figure 10 Particle size distribution (A) and Zeta potential distribution (B) of Astragalus membranaceus extracellular vesicle-derived lipid particles prepared in Example 2.

[0039] Figure 11 Disease activity index (DAI) changes of mice in each group in Example 2.

[0040] Figure 12 Colon length changes of mice in each group in Example 2. Wherein, Figure 12 A is the colon length photo of mice in each group, Figure 12 B is the colon length measurement result of mice in each group.

[0041] Figure 13 Organ coefficients and appearance changes of mice in each group in Example 2. Wherein, Figure 13 A is the spleen coefficient, Figure 13 B is the kidney coefficient, Figure 13C is the liver coefficient, Figure 13 D is a representative photo of the appearance of the spleen, kidney, and liver.

[0042] Figure 14 The results of pathological sections of the colon of mice in each group in Example 2 are shown in the following table. Figure 14 A is the normal group, Figure 14 B is the model group, Figure 14 C is the mesalazine group, Figure 14 D is the astragalus extracellular vesicle group, Figure 14 E is the astragalus extracellular vesicle lipid granule group. The scale bar in the figure is 50 pm. DETAILED DESCRIPTION

[0043] For the convenience of those skilled in the art, some components involved in the present application are further described below.

[0044] As used herein, the dosage form of the product of the present application is a powder, tablet, capsule, granule, or oral liquid. Preferably, the dosage form of the present application is a tablet or capsule.

[0045] As used herein, the present application can also comprise a "pharmaceutically, functionally, or health care acceptable carrier" in the product of the present application, which refers to a carrier that is conventional in the above technical field, selected from one or more of a filler, a binder, a disintegrant, a lubricant, a suspending agent, a wetting agent, a pigment, a flavoring agent, a solvent, and a surfactant.

[0046] The filler of the present application includes, but is not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, sugar powder, glucose, etc.; the lubricant includes, but is not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binder includes, but is not limited to, water, ethanol, starch paste, sugar syrup, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrant includes, but is not limited to, starch effervescent mixture, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agent includes, but is not limited to, polysaccharides such as acacia, agar, alginic acid, cellulose ether, and carboxymethyl chitin, etc.; the solvent includes, but is not limited to, water, balanced salt solution, etc.

[0047] Preferably, the product of the present application can be prepared into various solid oral preparations, liquid oral preparations, etc. Acceptable solid oral preparations include powder, ordinary tablet, dispersible tablet, enteric-coated tablet, granule, capsule, dripping pill, and powder, etc., and liquid oral preparations include oral liquid and emulsion, etc.

[0048] The above various dosage forms can be prepared according to conventional processes in the field of pharmaceutical preparations.

[0049] In the above described uses, the time of administration, the number of administrations, the frequency of administration, and the like of the various active ingredients need to be determined according to the actual conditions of the subject, which is within the skill of the person skilled in the art. For example, the therapeutic regimen for mice or rats can be applied to humans, and the effective dose of all active ingredients for humans can be converted from the effective dose for mice or rats, which is also easily achieved by the person skilled in the art.

[0050] The preferred embodiments of the present application are described below, and it should be understood that the preferred embodiments described herein are used only for illustration and explanation of the present application, and are not intended to limit the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be purchased through a regular channel.

[0051] Example 1: Preparation, identification, structural characterization of various extracellular vesicles, and pharmacodynamic study on a mouse colitis model

[0052] 1.1 Preparation of traditional Chinese medicine extracellular vesicles:

[0053] Preparation of Astragalus membranaceus extracellular vesicles (also known as extracellular vesicles): In this embodiment, PEG sedimentation method was used to obtain Astragalus membranaceus extracellular vesicles. Briefly, Astragalus membranaceus (from Dingxi, Gansu, three years old) was preliminarily washed with tap water at room temperature, and then rinsed with PBS solution for 3 times. The washed Astragalus membranaceus was cut into pieces with a thickness of about 5 mm, soaked with appropriate amount of PBS, broken in a cell disrupter for 3 min, filtered with a gauze to remove the Astragalus membranaceus residues, and the Astragalus membranaceus juice was collected. The filtered Astragalus membranaceus juice was centrifuged at 3,000g for 30 min to collect the supernatant, and then the collected supernatant was centrifuged at 5,000g for 40 min to collect the supernatant, and then the collected supernatant was centrifuged at 15,000g for 30 min, and then the supernatant was sedimented with 8% PEG6000 (containing 500 mM NaCl) overnight. The sedimented Astragalus membranaceus supernatant was centrifuged at 5,000g for 30 min to obtain the precipitate, which was resuspended with filtered PBS solution, and the pH was adjusted to neutral, and then Astragalus membranaceus extracellular vesicles were obtained.

[0054] Preparation of Schisandra chinensis extracellular vesicles: prepared according to the preparation procedure of Astragalus membranaceus extracellular vesicles.

[0055] Preparation of Shenling Baizhu Powder extracellular vesicles: prepared according to the preparation procedure of Astragalus membranaceus extracellular vesicles. Shenling Baizhu Powder prescription: lotus seed meat 50 g, yiyi 50 g, amomum 50 g, platycodon 50 g, white kidney bean 75 g, white hoelen 100 g, ginseng 100 g, fried licorice 100 g, white atractylodes 100 g, Chinese yam 100 g.

[0056] 1.2 Identification of extracellular vesicles from traditional Chinese medicine

[0057] (1) Transmission electron microscopy (TEM) structural characterization

[0058] Take 20 μL of the Astragalus extracellular vesicle suspension prepared in Section 1.1 above and drop it onto a copper grid with a carbon support membrane. Let it sit at room temperature for 5 minutes to allow for natural adsorption. Remove excess liquid with filter paper. Then, add 20 μL of a 2% phosphotungstic acid solution to the copper grid. Stain the membrane for 3 minutes. Remove excess liquid with filter paper. Allow the grid to air dry under an incandescent light. Observe the morphology of the extracellular vesicles using a transmission electron microscope.

[0059] The general state under the transmission electron microscope is saucer-shaped or concave round cake-shaped. Figure 1 As shown, the obtained Astragalus extracellular vesicles were observed under transmission electron microscopy to have a clear structure, mostly in the shape of concave round cakes, with a relatively clean background, and a size distribution in the range of 50-150nm.

[0060] (2) Characterization of extracellular vesicles by nanoflow cytometry

[0061] Nanoflow cytometry (FCM) can achieve highly sensitive, highly selective, and high-throughput detection of the particle size and distribution, particle concentration, and biochemical properties of single nanoparticles (7-1000nm) as well as natural biological nanoparticles such as extracellular vesicles (exosomes), viruses, bacteria, and subcellular structures.

[0062] Analysis of the test results showed a unimodal distribution of particles within the sample. The average particle size distribution of extracellular vesicles from Astragalus membranaceus was 81.13±23.74nm, similar to the size observed by TEM. The average particle size distribution of extracellular vesicles from Schisandra chinensis was 83.82±17.70nm. The average particle size distribution of extracellular vesicles from Shenling Baizhu Powder was 70.96±15.74nm.

[0063] The concentration of Astragalus extracellular vesicles was 1.08 x 10 11 / mL. The concentration of Schisandra chinensis extracellular vesicles is 7.69x 10 10 The extracellular vesicle concentration of Shenling Baizhu Powder is 8.47 x 10 10 The peak distribution results of extracellular vesicle size are as follows: Figure 2 shown.

[0064] (3) Extracellular vesicle zeta potential analysis

[0065] The zeta potential of the extracellular vesicles of Astragalus prepared in section 1.1 above was analyzed. The results of the Malvern laser particle size analyzer showed that the zeta potential of the extracellular vesicles of Astragalus was unimodal, with an average potential distribution of -34.2±7.09mV. Figure 3The results showed that the obtained extracellular vesicles of Astragalus membranaceus were negatively charged on the surface and the system was relatively stable.

[0066] 1.3 Pharmacodynamic study on mouse colitis model

[0067] To explore the therapeutic effect of the extracellular vesicles prepared in the above 1.1 part of Example 1 on DSS-induced mouse colitis. The DSS-induced mouse UC model is one of the most widely used animal models for studying inflammatory bowel disease. The symptoms and histological changes of this model are highly similar to human UC, and can be used to study the mechanisms of acute and chronic UC and the pharmacodynamic effects of drugs. In Example 1, the DSS-induced UC mouse model was used to analyze the effects of Astragalus membranaceus and other traditional Chinese medicine extracellular vesicles on the intestinal immune response and barrier function of mice. Meanwhile, the clinically commonly used drug mesalazine was used as a positive control to develop a new safe and effective method for treating UC.

[0068] 1.3.1 Establishment and evaluation of animal model

[0069] The DSS-induced UC mouse model was established according to the method of Cooper et al., and the classic C57BL / 6J mice (Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were used to freely drink DSS solution for 7 days to establish the model, and the intervention effect was observed by gavage. The specific operation is as follows: 6-8 week-old C57BL / 6J male mice were selected, ear tagged, and adaptively fed for seven days; on the eighth day, all mice except the normal group were allowed to drink 3% (w / v) DSS solution for 7 days, and the inducing drug solution was replaced every two days to establish the experimental UC mouse model; at the same time, 0.2 mL of the corresponding drug intervention was administered to each mouse by gavage every day, and 0.2 mL of PBS was administered to each mouse in the normal group by gavage every day as a control. The food and water intake of the mice was recorded every day, the body weight of the mice was recorded at a fixed time every day, the stool characteristics of the mice were observed, and the degree of occult blood in the mice was determined according to the method of the occult blood kit and recorded, and finally the disease activity index (Disease activity index, DAI) was calculated. On the 15th day, the 3% DSS solution was stopped and replaced with pure water, and the mice were fasted for one day. On the 16th day, the body weight of the mice was recorded, the mice were anesthetized with 10% chloral hydrate, and the mice were sacrificed by decapitation. The liver, spleen, kidney and colon tissues were collected. The fresh liver, spleen and kidney were weighed, and the organ coefficients were calculated. The length of the mouse colon was measured, and the data was recorded. The colon was washed with physiological saline and dried with a test paper. The colon tissue at the second centimeter of the distal colon was taken and immersed in formalin, gently inverted, and quickly fixed. The remaining colon was quickly transferred to a -80°C refrigerator for storage.

[0070] Drug grouping: normal group, model group, mesalazine group, Shenling Baizhu Powder extracellular vesicles group, Astragalus membranaceus extracellular vesicles group, Schisandra chinensis extracellular vesicles group. Intragastric administration during modeling. The amount of each group is shown below. The volume of administration is 0.2 mL.

[0071] Normal group: 0.2 mL of PBS per day

[0072] Model group: 0.2 mL of PBS per day

[0073] Mesalazine group: 0.52 g / kg / day (PBS suspension) (positive control)

[0074] Shenling Baizhu Powder extracellular vesicles group: about 5 x 10 9 particles per day

[0075] Astragalus membranaceus extracellular vesicles group: about 5 x 10 9 particles per day

[0076] Schisandra chinensis extracellular vesicles group: about 5 x 10 9 particles per day.

[0077] Statistical analysis of experimental data: data analysis, statistical analysis and picture processing were performed using GraphPad Prism 8.0 software. The measurement data was expressed as mean ± standard deviation (Mean ± SD), and the data between groups was analyzed by one-way ANOVA, and * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 had statistical difference.

[0078] 1.3.2 Analysis of animal experiment results

[0079] (1) DAI score of mice

[0080] The main function of the colon is to absorb water and electrolytes, form, store and excrete feces. If the colon produces inflammation and functional disorder, it will cause the mouse to lose weight, increase fecal water, produce loose stools, damage the colon barrier, and cause intestinal edema, bleeding, and produce bloody stools. The above three factors are combined to reflect the severity of mouse colitis in the form of DAI score, to evaluate the disease and investigate the therapeutic effect of the intervention drug.

[0081] DAI refers to the incidence index of mouse disease, and the higher the score, the stronger the inflammation.

[0082] DAI score = (body weight loss% + fecal character + fecal occult blood) / 3, wherein the body weight change rate (%) = (body weight on the day / g) / (initial body weight / g) x 100. The mouse disease activity index was calculated according to the scores of the three of body weight loss%, fecal character, and fecal occult blood (Table 1).

[0083] Table 1: Mouse disease activity index (DAI)

[0084]

[0085] The results of this experiment showed that DAI was model group > Schisandra chinensis exosome group > Shenling Baizhu San exosome group > mesalazine group > Astragalus membranaceus exosome group > normal group; and there was a significant difference between the Astragalus membranaceus exosome group and the model group, and no significant difference with the normal group, while the screened Schisandra chinensis exosome group and Shenling Baizhu San exosome group had no significant difference with the model group. Figure 4

[0086] (2) Colon length of mice

[0087] The colon length is also a reference index reflecting the severity of colitis. With the increase of inflammation, the colon of mice will gradually shorten.

[0088] The colon length in this experiment: normal group > Astragalus membranaceus exosome group > mesalazine group > Schisandra chinensis exosome group > Shenling Baizhu San exosome group > model group. The colon length of the Astragalus membranaceus exosome group had no significant difference with the normal group, and had a significant difference with the model group. The inflammation caused by DSS in mice was significantly reduced after administration of Astragalus membranaceus exosome. The other two exosome groups had no such effect. Figure 5

[0089] (3) Changes in organ coefficients of mice

[0090] The organ coefficient is the ratio of the mass of each organ of the experimental animal to the body weight, which is a standard commonly used in toxicological experiments. It is relatively stable under normal physiological conditions, but changes when the body has inflammation or immune response. An increase in organ coefficient indicates that the organ has edema, hyperemia, etc.; on the contrary, it represents atrophic changes such as organ atrophy. The occurrence of colitis is generally accompanied by the production of splenomegaly, and the increase of spleen coefficient.

[0091] The spleen coefficient of mice is shown in Figure 6 A. The spleen coefficient of the model group without drug intervention was significantly increased after ingesting DSS, indicating that the mouse spleen was damaged. After intervention with the control drug mesalazine or Astragalus membranaceus exosome and Shenling Baizhu San exosome, the spleen coefficient of mice was significantly reduced, but the intervention of Schisandra chinensis exosome actually increased the spleen coefficient of mice. Figure 6 ​​As shown in FIG. B, the decrease in kidney coefficient indicates the atrophy of the kidney, which may be due to the renal toxicity of the administered drug. In this experiment, the kidney coefficient was: normal group > DSS group > Huangqi extracellular vesicle group > Wuweizi extracellular vesicle group > Shenlingbaizhu extracellular vesicle group > mesalazine group. The kidney coefficient of the mesalazine group was the lowest, which was significantly different from that of the normal group, indicating that the kidney of the mouse was damaged. There was no significant difference between the other groups, which may be related to the renal toxicity of mesalazine itself.

[0092] Therefore, Huangqi extracellular vesicles can alleviate the increase in spleen coefficient induced by DSS; at the same time, they do not have the renal toxicity of mesalazine.

[0093] In summary, in Example 1, the structure of Huangqi extracellular vesicles was comprehensively characterized to confirm the characteristics of the extracellular vesicles. Then, according to the conventional method, 3% DSS solution was used to induce mouse colitis, mesalazine intervention was used as a positive control, and Huangqi extracellular vesicles and other traditional Chinese medicine extracellular vesicles were administered for intervention. The results of the observation of various inflammatory indicators showed that Huangqi extracellular vesicles had a therapeutic effect on DSS-induced mouse UC. The DAI score results showed that Huangqi extracellular vesicles significantly reduced the trend of DAI score of the model group mice; the colon length results showed that Huangqi extracellular vesicles had a significant effect on alleviating the shortening of the colon of the model mice; the spleen coefficient results in the organ coefficient showed that Huangqi extracellular vesicles had an effect on alleviating the increase in the spleen coefficient of the mice, while having no significant effect on the kidney. The effects of the extracellular vesicles of the other several traditional Chinese medicines were much weaker. This example showed that Huangqi extracellular vesicles had an effect on alleviating DSS-induced mouse UC.

[0094] Example 2: Pharmacodynamic study of Huangqi extracellular vesicles prepared by improved method and Huangqi extracellular vesicle-derived lipid particles on mouse colitis model

[0095] Further explore the effect of Huangqi extracellular vesicles prepared by improved method on alleviating DSS-induced mouse colitis, and explore the effect of Huangqi extracellular vesicle-derived lipid particles on mouse colitis.

[0096] 2.1 Preparation of Huangqi extracellular vesicles:

[0097] The extraction method of extracellular vesicles was improved by using differential-ultracentrifugation method combined with sucrose density gradient centrifugation to obtain purer Huangqi extracellular vesicles.

[0098] Take Astragalus membranaceus, wash it preliminarily with tap water at room temperature, and then rinse it with PBS solution three times. Cut the washed Astragalus membranaceus into slices with a thickness of about 5 mm, add an appropriate amount of PBS to soak, and after soaking fully, crush it in a wall-breaking machine for 3 minutes, filter it with a gauze, remove the Astragalus membranaceus residue, and collect the Astragalus membranaceus juice. Under 4°C conditions, the filtered Astragalus membranaceus juice was centrifuged at 3,000g×30min to collect the supernatant, and then the collected supernatant was centrifuged at 5,000g×40min to collect the supernatant, and then the collected supernatant was centrifuged at 15,000g×30min to collect the supernatant, and then the collected supernatant was centrifuged at 150,000×70min to obtain a precipitate. Resuspend the precipitate with filtered and sterilized PBS solution to preliminarily obtain Astragalus membranaceus extracellular vesicles, and then obtain relatively pure Astragalus membranaceus extracellular vesicles by sucrose density gradient centrifugation at 20,000g×2h. The process is as follows. Figure 7 shown.

[0099] 2.2 Preparation of Astragalus Extracellular Vesicle Lipid Nanoparticles:

[0100] Take 4 mL of methanol and 2 mL of chloroform in a clean glass test tube, add 1 mL (containing 8.46 x 10 10 The Astragalus extracellular vesicle suspension prepared in Section 2.1 above was thoroughly vortexed for 1 minute, and 2 mL of chloroform and 2 mL of purified water were added, and the mixture was thoroughly vortexed for 1 minute. Subsequently, the mixture was centrifuged at 2300 rpm for 10 minutes. The lower organic phase was removed using a glass pipette and transferred to a new glass test tube, which was then dried under nitrogen. 600 μL of PBS solution was added to the dried glass test tube, and the mixture was sonicated in a water bath for 5 minutes. The mixture was then filtered through a 0.45 μm filter to obtain lipid nanoparticles derived from Astragalus extracellular vesicles.

[0101] 2.3 Identification of Astragalus Extracellular Vesicles and Astragalus Extracellular Vesicle Lipid Nanoparticles

[0102] (1) Transmission electron microscopy (TEM) structural characterization

[0103] The experimental method is shown in Section 1.2 of Example 1. Figure 8 As shown, the results showed that the obtained Astragalus extracellular vesicles were double-membrane structures, mostly in the shape of concave round cakes, with a clean background, and a size distribution in the range of 50-150nm.

[0104] (2) Characterization of Astragalus extracellular vesicles by nanoflow cytometry

[0105] The Astragalus extracellular vesicle sample obtained in section 2.1 above was diluted and tested on an instrument.

[0106] The results of the detection analysis showed that the particles in the sample were unimodal distribution, and the average particle size distribution range of the astragalus cell extracellular vesicles was 80.95±15.84 nm. The concentration of the astragalus cell extracellular vesicles was 8.46x 10 10 The particle size peak distribution of the astragalus cell extracellular vesicles is shown in Figure 9 .

[0107] (3) Appearance of astragalus cell extracellular vesicle lipid nanoparticle suspension

[0108] The astragalus cell extracellular vesicle lipid particle suspension prepared from the lipid components extracted from the astragalus cell extracellular vesicles was colorless and transparent under visual observation, and no precipitate or suspended particles were visible to the naked eye, which was similar to the appearance of the lipid particles prepared in the literature, which was a colorless transparent liquid.

[0109] (4) Analysis of astragalus cell extracellular vesicle lipid nanoparticle particle size and Zeta potential

[0110] The results of the Malvern laser particle size analyzer analysis showed that, as shown in Figure 10 A, the astragalus cell extracellular vesicle-derived lipid particle size was unimodal distribution, and the average particle size distribution range was 266.5 nm. As shown in Figure 10 B, the Zeta potential of the astragalus cell extracellular vesicle-derived lipid particles was unimodal distribution, and the average Zeta potential was -51.2±7.74 mV. Compared with the astragalus cell extracellular vesicles, the Zeta potential of the lipid particles was larger, indicating that the dispersion and stability of the system were stronger.

[0111] 2.4 Pharmacodynamic study on a mouse colitis model

[0112] 2.4.1 Establishment and evaluation of animal models

[0113] The animal model preparation method, experimental data representation and statistical analysis method are described in section 1.3.1 of Example 1.

[0114] After one week of adaptive feeding, the mice were randomly divided into 5 groups (n=8): normal group, model group, mesalazine group, astragalus cell extracellular vesicle group, and astragalus cell extracellular vesicle lipid particle group. Except for the normal group of mice, the other groups of mice freely drank 3% DSS solution to establish an experimental UC mouse model. The complete mesalazine enteric-coated tablets were ground into powder, weighed, added to PBS to prepare a suspension, and administered orally with 0.2 mL of mesalazine at a dose of 0.52 g / Kg per day in the morning; the astragalus cell extracellular vesicle group was administered orally with 0.2 mL of astragalus cell extracellular vesicle suspension at a dose of 1x 10 10 particles per mouse per day in the morning; the astragalus cell extracellular vesicle lipid particle group was administered orally with 0.2 mL of astragalus cell extracellular vesicle-derived lipid particles at the same dose, once a day for 7 consecutive days. The normal group and the model group were administered with an equal volume of PBS solution every day.

[0115] 2.4.2 Experimental result analysis

[0116] (1) DAI score

[0117] According to the standard of Table 1 in Example 1, the DAI of each group of mice within 7 days of drug intervention was calculated and the change curve was drawn. As shown in Figure 11 , the DAI score of the normal group was the lowest and was at the normal level; the DAI score of the model group mice was the highest, most of the mice had rectal bleeding and loose stool mucus sticking to the anus on the seventh day, and the mice had obvious colitis; under the intervention of Astragalus membranaceus extracellular vesicles and their lipid particles, the DAI score of the model mice was significantly reduced compared with the model group, which significantly relieved the UC of the mice, and the DAI score was similar to that of the positive control drug mesalazine, and there was no significant difference in the DAI score among the three groups. The results showed that Astragalus membranaceus extracellular vesicles and their lipid particles had obvious effect on relieving UC of mice, and the lipid component might be an important component of Astragalus membranaceus extracellular vesicles to play a role in relieving inflammation.

[0118] (2) Effect on colon length

[0119] Under the stimulation of modeling drugs, the colon of colitis mice would be significantly shortened, and the intestinal contents would not be shaped. The results are shown in Figure 12 , compared with the normal group, the colon of the model group mice appeared obvious edema, and the colon was significantly shortened. After the intervention of Astragalus membranaceus extracellular vesicles and their lipid particles, the colon length of mice was significantly increased compared with the model group. The effect was similar to that of the mesalazine group. This also proved that Astragalus membranaceus extracellular vesicles and their lipid particles could significantly relieve the symptoms of colon shortening in colitis mice.

[0120] (3) Effect on organ coefficient

[0121] The occurrence of colitis is generally accompanied by the occurrence of splenomegaly and the increase of spleen coefficient. The spleen coefficient of mice is shown in Figure 13 A. After ingesting DSS, the spleen coefficient of the model group without drug intervention was significantly increased, indicating that the mouse spleen was damaged. After the intervention of the control drug mesalazine or Astragalus membranaceus extracellular vesicles and their lipid particles, the spleen coefficient of mice was significantly reduced, and the effect of Astragalus membranaceus extracellular vesicles and their lipid particles on reducing spleen damage was the most obvious. The kidney coefficient of mice is shown in Figure 13 B. The kidney coefficient of mice in the mesalazine group was the lowest, indicating that the kidney of mice was damaged, and there was no significant difference between the other groups, which might be related to the renal toxicity of mesalazine itself; the liver coefficient is shown in Figure 13 C. There was no significant difference in the liver coefficient among the mice in each group. In summary, Astragalus membranaceus extracellular vesicles and their lipid particles can relieve the increase of DSS-induced spleen coefficient; at the same time, they do not have the renal toxicity of mesalazine.

[0122] Representative photos of the appearance of the spleen, kidney and liver of mice in each group are as follows:Figure 13 D as shown.

[0123] (4) Colonic pathological injury in mice

[0124] H&E staining and microscopic observation of mouse colon tissue sections can directly show the damage to the colon tissue and the degree of inflammatory cell infiltration. The results of pathological analysis of mouse colon tissue are shown in Figure 14 As shown in A-14E, compared with the normal group, DSS greatly damaged the integrity of the colon mucosa, with severe inflammatory cell infiltration and ulceration inside the colon, severe loss of intestinal villi, and most of the goblet cells disappeared. Mesalazine significantly reduced the inflammatory cell infiltration of the colon tissue, and the number of disappeared goblet cells was significantly reduced, but the improvement effect on the damage to the colon mucosa was not ideal. Astragalus extracellular vesicles significantly reduced the inflammatory cell infiltration inside the colon, and the morphology and structure of the intestinal villi were still complete, protecting the integrity of the colon mucosa. Compared with the DSS group, the astragalus extracellular vesicle lipid particles significantly reduced the inflammatory cell infiltration inside the colon, the number of goblet cells was large, and the colon mucosa was intact.

[0125] The structure of the intestinal epithelium is the basis for maintaining intestinal homeostasis. The intestine is inhabited by a large number of complex microorganisms. The results of this experiment show that DSS severely damaged the colon epithelial monolayer structure, which cannot effectively resist the invasion of harmful substances from the outside world and the influence on intestinal cells, causing the activation of antigen-presenting cells, thereby leading to severe inflammatory cell infiltration. Astragalus extracellular vesicles and their lipid particles effectively alleviate the damage to the colon mucosa and inhibit inflammatory cell infiltration. From this analysis, the protection of astragalus extracellular vesicles and their lipid particles to the intestinal barrier may be the basis for improving DSS-induced diarrhea and hematochezia.

[0126] In summary, according to the common method, the mice were induced with 3% DSS solution to induce colitis, mesalazine intervention was used as a positive control, and Astragalus membranaceus extracellular vesicles were used to observe the inflammatory indicators to explore the therapeutic effect of Astragalus membranaceus extracellular vesicles on DSS-induced UC in mice. At the same time, Astragalus membranaceus extracellular vesicle lipid granules were given to inflammatory mice to observe the inflammatory indicators to observe whether the Astragalus membranaceus extracellular vesicle lipid granules were part of the Astragalus membranaceus extracellular vesicles. First, the daily DAI of mice was calculated to characterize the colitis of mice, and the higher the score, the stronger the inflammation of mice. Second, the colon length of mice was measured, and the greater the degree of colon shortening, the stronger the inflammation, and then the mouse colon tissue pathological sections were prepared and H&E staining was used to observe the degree of pathological changes in mouse colon tissue. The disappearance of crypts and goblet cells in colon tissue, destruction of glands, and incomplete upper skin indicated that the mouse colon had UC. The DAI score results showed that Astragalus membranaceus extracellular vesicles and their lipid granules significantly reduced the DAI score of model mice from 3.0 points to 1.5 points; the colon length results showed that Astragalus membranaceus extracellular vesicles and their lipid granules significantly relieved the colon shortening of model mice; the spleen coefficient results in the organ coefficient showed that Astragalus membranaceus extracellular vesicles and their lipid granules had the effect of relieving the increase of mouse spleen coefficient, but had no obvious effect on the kidney and liver; the colon pathological sections showed that Astragalus membranaceus extracellular vesicles and their lipid granules could significantly relieve the disappearance of colon goblet cells and crypt cells and the absence of colon glands and epithelial damage caused by DSS.

[0127] Therefore, Astragalus membranaceus extracellular vesicles and their lipid granules have the effect of relieving DSS-induced UC in mice, and their lipid components may be one of the effective parts of their pharmacodynamic effects.

[0128] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. The use of Astragalus membranaceus extracellular vesicle lipid particles in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The astragalus extracellular vesicle lipid particle is nanoscale, and a preparation method of the astragalus extracellular vesicle lipid particle comprises the following steps: taking a mixed solution of methanol and chloroform in a clean glass test tube, adding an astragalus extracellular vesicle suspension, fully vortexing for 1 min, adding a mixed solution of chloroform and pure water, fully vortexing for 1 min, then centrifuging, taking the lower organic phase to a new glass test tube by using a glass transfer tube, blowing dry by nitrogen, adding a PBS solution to the dried glass test tube, water bath ultrasonic for 5 min, filtering through a 0.45 μm filter, and obtaining astragalus extracellular vesicle lipid particles.

2. Use according to claim 1, characterized in that: The drug is an oral preparation or a rectal administration preparation.

3. Use according to claim 2, characterized in that: The oral preparation is selected from an oral liquid, a tablet, a powder, a capsule or a granule.

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