Kudzuvine root vesicle-like nanoparticles as well as extraction method and application thereof

Abstract

The invention discloses kudzu vine root vesicle-like nanoparticles as well as an extraction method and application thereof, and belongs to the technical field of biological medicines. The method comprises the following steps: crushing dry radix puerariae, adding ultrapure water according to a certain proportion, and boiling for 45 minutes to prepare radix puerariae decoction; carrying out gradient centrifugation at the temperature of 4-8 DEG C by 1000g-12000g to remove impurities, and taking supernate, so as to obtain crude extract of the kudzu vine root vesicle-like nanoparticles; then carrying out 150000-170000 g ultracentrifugation at the temperature of 4-8 DEG C, so as to obtain a crude extract of the kudzu vine root vesicle-like nanoparticles; the crude extract is centrifuged for 2-5 min at the temperature of 4-8 DEG C at the speed of 4000-6000 rpm, supernate is taken, and after the supernate is filtered through a 0.22-micron filter membrane, the kudzu vine root vesicle-like nano-particles are obtained. The invention provides the method for extracting the vesicle-like nanoparticles in the radix puerariae decoction, and the radix puerariae vesicle-like nanoparticles extracted by the method can be remarkably enriched in the inflammatory intestinal tract part, so that efficient and safe treatment of ulcerative colitis is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an extraction method of kudzu root vesicle-like nanoparticles and application thereof. BACKGROUND

[0002] Kudzu root is a traditional Chinese medicine, which is the dried root of Pueraria lobata (Willd.) Ohwi, and has a long history of application. Kudzu root has health care functions of being both medicine and food. Kudzu root contains various active ingredients, which have pharmacological effects including improving blood circulation, reducing blood pressure, reducing blood sugar, anti-tumor, immune defense, anti-inflammatory and anti-oxidation. Due to its strong and complex pharmacological effects, kudzu root has shown good treatment potential for various diseases including inflammatory bowel disease (IBD). Inflammatory bowel disease is a common continuous inflammatory disease of the digestive system, and ulcerative colitis (UC) is a typical representative thereof. UC usually affects the rectum in a continuous manner, affects part or the entire colon, and spreads to the surrounding appendix, causes inflammation of the intestinal mucosa or submucosal layer, is accompanied by crypt lesions and abscesses, and has different degrees of systemic symptoms such as abdominal pain, diarrhea, and mucous pus blood stool. At present, the incidence of IBD is increasing year by year, which has a huge impact on public health around the world.

[0003] At present, kudzu root has been proved to have a therapeutic effect on UC, and the exploration of active ingredients playing a therapeutic effect on kudzu root focuses on small molecule compounds such as puerarin. Puerarin is limited in clinical application due to its poor water solubility, in vivo degradation, fast in vivo elimination, and low oral bioavailability. Therefore, it is of great significance to develop new active ingredients contained in kudzu root. SUMMARY

[0004] Plant derived vesicle-like nanoparticles (PDVLNs) are a new type of active ingredient found in plants, which is a bioactive substance with a phospholipid bilayer, contains proteins, lipids, DNA, RNA, phytochemicals and other ingredients, and plays an important role in intercellular signal transduction, cargo transport and disease diagnosis. PDVLNs have low immunogenicity, are easy to obtain and low in cost, and therefore are safer and more economical as therapeutic agents and drug carriers. Humans intake various PDVLNs in daily diet, and researches have shown that these nanoparticles can enter the human system and deliver effector molecules to cells, thereby regulating cell signaling pathways and playing an important role in treating diseases and improving human health. At present, there is no evidence that PDVLNs from kudzu root have a therapeutic effect on UC.

[0005] Therefore, the present application aims to provide a Pueraria lobata vesicle-like nanoparticle and an extraction method and application thereof, and the Pueraria lobata vesicle-like nanoparticle extracted by the present application has good therapeutic effect on ulcerative colitis.

[0006] To achieve the above-mentioned object, the technical scheme of the present application is as follows:

[0007] In the first aspect of the present application, an extraction method of Pueraria lobata vesicle-like nanoparticle is provided, comprising the following steps:

[0008] S1, dry Pueraria lobata is crushed, and is decocted in ultrapure water for 30-60 min, and after cooling, the decoction is filtered by gauze extrusion to obtain a Pueraria lobata decoction;

[0009] S2, the Pueraria lobata decoction of S1 is sequentially subjected to gradient centrifugation at 1000-12000 g at 4-8℃ for 10-50 min, and the supernatant is taken to obtain a crude extract of Pueraria lobata vesicle-like nanoparticle;

[0010] S3, the crude extract of S2 is subjected to ultracentrifugation at 150000-170000 g at 4-8℃, the supernatant is discarded, and the bottom precipitate is resuspended with phosphate to obtain a crude extract of Pueraria lobata vesicle-like nanoparticle;

[0011] S4, the obtained crude extract is subjected to low-speed centrifugation at 4000-6000 rpm at 4-8℃, the supernatant is taken, and the Pueraria lobata vesicle-like nanoparticle is obtained after filtration through a filter membrane.

[0012] Further, in step S1,

[0013] The crushing time is 20-40 s;

[0014] When the ultrapure water is added for decoction, the w / v of the dry Pueraria lobata powder and the ultrapure water is 1:6-1:10, the decoction time is 45 min, and the gauze aperture is 100 mesh.

[0015] Further, in step S2,

[0016] The gradient centrifugation is specifically 1000-1500 g for 10-15 min, 3000-5000 g for 20-30 min, and 10000-12000 g for 30-50 min.

[0017] Further, in step S3, the ultracentrifugation is 150000 g ultracentrifugation for 70-90 min.

[0018] Further, in step S4, the low-speed centrifugation is 5000 rpm centrifugation for 2-5 min, and the filter membrane aperture is 0.22 μm.

[0019] In a second aspect of the present application, the Puerariae radix vesicle-like nanoparticles prepared by any of the above-mentioned methods are provided.

[0020] Further, the particle size of the Puerariae radix vesicle-like nanoparticles is 100-300 nm, and the electric potential of the Puerariae radix vesicle-like nanoparticles is -3 to -9 mv.

[0021] In a third aspect of the present application, the Puerariae radix vesicle-like nanoparticles are used for preparing a drug for treating colitis.

[0022] Further, the colitis is ulcerative colitis.

[0023] The present application also provides a pharmaceutical preparation comprising the Puerariae radix vesicle-like nanoparticles and a pharmaceutically acceptable carrier, wherein the Puerariae radix vesicle-like nanoparticles serve as active ingredients.

[0024] Further, the pharmaceutical preparation is in the form of oral preparations, including oral liquids, tablets, capsules, granules, etc.

[0025] Further, the carrier of the pharmaceutical preparation is a common carrier in the pharmaceutical field, including solvents, solubilizers, coating materials, preservatives, diluents, stabilizers, binders, flavoring agents, coloring agents, etc.

[0026] The present application has at least the following advantages:

[0027] (1) In the present application, the Puerariae radix vesicle-like nanoparticles, as a new active ingredient in Puerariae radix, are nanostructures carrying proteins, lipids and other active substances, which improve the bioavailability of the effective ingredients in Puerariae radix and can treat ulcerative colitis by relieving intestinal inflammation, restoring intestinal barrier and balancing intestinal microecology; and the Puerariae radix vesicle-like nanoparticles have good gastrointestinal stability and inflammation site retention, so they can be used as oral preparations, greatly improving patient compliance.

[0028] (2) The present application provides an extraction method of Puerariae radix vesicle-like nanoparticles, and the Puerariae radix is widely available, and the extraction process is simple, so the Puerariae radix vesicle-like nanoparticles are easy to obtain and have low cost.

[0029] (3) The Puerariae radix vesicle-like nanoparticles extracted in the present application have superior biocompatibility, no obvious toxicity to healthy tissue cells, low immunogenicity and low risk, and are suitable for commercial production.

[0030] (4) The Puerariae radix vesicle-like nanoparticles extracted in the present application provide a new choice for the treatment of ulcerative colitis.

[0031] (5) The stable vesicle-like nanoparticles are extracted from the high-temperature decocted Puerariae radix decoction, which also provides a reference for the subsequent study of drying plant vesicle-like nanoparticles.

[0032] (6) The present application provides a new idea for the exploration of active substances for treating ulcerative colitis in traditional Chinese medicine, promotes the integration of traditional Chinese medicine and modern medicine, and provides a new route for the use of traditional Chinese medicinal materials. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is an extraction schematic diagram of the Puerariae radix vesicle-like nanoparticles in Example 1.

[0034] Figure 2 It is a transmission electron microscope image of the Puerariae radix vesicle-like nanoparticles in Example 1, and the scale is 100 nm.

[0035] Figure 3 It is the nanoparticle tracking analysis result of the Puerariae radix vesicle-like nanoparticles in Example 1.

[0036] Figure 4 It is the Zeta potential diagram of the Puerariae radix vesicle-like nanoparticles in Example 1.

[0037] Figure 5 It is the transmission electron microscope observation result of the Puerariae radix vesicle-like nanoparticles in Example 1 after digestion in simulated gastric juice and simulated intestinal tract.

[0038] Figure 6 It is the lipid group analysis result of the Puerariae radix vesicle-like nanoparticles in Example 1, wherein A is the lipid composition analysis, and B is the KEGG analysis of lipids.

[0039] Figure 7 It is the quantitative analysis result of puerarin in the Puerariae radix vesicle-like nanoparticles in Example 1, wherein A is the standard curve of puerarin liquid chromatography, and B is the quantitative result of puerarin after bringing in the standard curve.

[0040] Figure 8 It is the mouse weight change diagram after the Puerariae radix vesicle-like nanoparticles are used for treating ulcerative colitis in Example 2.

[0041] Figure 9 It is the mouse disease activity index score diagram after the Puerariae radix vesicle-like nanoparticles are used for treating ulcerative colitis in Example 2.

[0042] Figure 10 It is the mouse colon length statistics after the Puerariae radix vesicle-like nanoparticles are used for treating ulcerative colitis in Example 2.

[0043] Figure 11Representative pictures of H&E staining of colon of mice after treatment with P. multiflora vesicle-like nanoparticles for ulcerative colitis in Example 2. Scale bar, 50 μm. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.

[0045] In addition, the technical solutions in the embodiments of the present application can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize the combination. When the combination of technical solutions appears to be contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the protection scope of the present application.

[0046] Unless otherwise specified, the experimental methods used in the present application are conventional methods, and the reagents and equipment can be obtained from commercial channels.

[0047] The technical solutions proposed in the present application will be specifically described below through specific embodiments:

[0048] Example 1: Extraction and characterization of P. multiflora vesicle-like nanoparticles

[0049] I. Extraction of P. multiflora vesicle-like nanoparticles

[0050] Step one, dry P. multiflora roots were placed in a pulverizer and pulverized for 30 s to obtain particles with a size of 50 mesh. According to a ratio of w / v of 1:6, ultrapure water was added and decocted for 45 min. After cooling, the P. multiflora decoction was obtained by filtering and squeezing using 100-mesh gauze.

[0051] Step two, the P. multiflora decoction was gradient centrifuged at 4℃ to remove dead cells, cell debris and large vesicle-like nanoparticles. The centrifugation steps were as follows: 1000g for 10 min, 3000g for 20 min and 10000g for 40 min. The supernatant was taken to obtain the crude extract of P. multiflora vesicle-like nanoparticles.

[0052] Step three, the crude extract of P. multiflora vesicle-like nanoparticles was ultracentrifuged at 150000g for 70 min at 4℃. The supernatant was discarded, and the bottom precipitate was resuspended with a phosphate buffer to obtain the crude extract of P. multiflora vesicle-like nanoparticles.

[0053] Step four, the crude extract of P. multiflora vesicle-like nanoparticles was centrifuged at 5000rpm for 2 min at 4℃ to remove the agglomerated particles. The supernatant was filtered through a 0.22-μm filter membrane to obtain stable and uniform P. multiflora vesicle-like nanoparticles.

[0054] The obtained Puerariae radix vesicle-like nanoparticles were stored at -80°C.

[0055] II. Characterization of Puerariae radix vesicle-like nanoparticles

[0056] 1. Transmission electron microscope observation of its morphology: Puerariae radix vesicle-like nanoparticles were diluted to 4 mg / mL, about 20 μL sample was dropped on the copper grid, and Puerariae radix vesicle-like nanoparticles were allowed to settle for 3-5 min, and then the excess water was wiped off. Then it was placed on 2% phosphotungstic acid staining solution for 1-2 min, and the excess staining solution was wiped off, and it was naturally air-dried. The copper grid was imaged using a transmission electron microscope, and a low-power lens was used for observation to determine the area to be imaged, and then a high-power lens was used to collect images.

[0057] From the above, Figure 2 it can be seen that Puerariae radix vesicle-like nanoparticles are cup-shaped structures under the microscope, have obvious lipid bilayers, are similar to animal exosome structures, and have a size of 100-200 nm.

[0058] 2. Nanoparticle tracking analysis for detecting the particle size distribution: Puerariae radix vesicle-like nanoparticles were diluted to 1 mg / mL, and Zetaview-PMX120-Z was used for nanoparticle tracking analysis of Puerariae radix vesicle-like nanoparticles to determine the particle size and particle number distribution graph.

[0059] From the above, Figure 3 it can be seen that the average particle size of Puerariae radix vesicle-like nanoparticles is 198.3 nm, which is consistent with the transmission electron microscope observation result, and there are about 1.5 x 10 11 nanoparticles in 1 mg / mL of Puerariae radix vesicle-like nanoparticles.

[0060] 3. Potential analysis of Puerariae radix vesicle-like nanoparticles: Puerariae radix vesicle-like nanoparticles were diluted to 1 mg / mL, and the sample cell was added, the electrode was inserted into the sample cell, and connected with the instrument, and the nanoparticle size and Zeta potential analyzer were used for potential determination.

[0061] From the above, Figure 4 it can be seen that the potential of Puerariae radix vesicle-like nanoparticles in phosphate buffer is -5.86 mv, which is consistent with the theory that the lipid bilayer is negatively charged.

[0062] 4. Gastrointestinal Stability Analysis of Kudzu Root Vesicle-like Nanoparticles: Simulated gastric and intestinal fluids were prepared according to the United States Pharmacopeia. The formulations of the simulated gastric and intestinal fluids were: 80 mM HCl, 35 mM NaCl, 0.3% pepsin, pH 1.2 and 15 mM NaOH, 50 mM KH2PO4, 1% trypsin, pH 6.8, respectively. Kudzu root vesicle-like nanoparticles were dispersed in phosphate buffer, simulated gastric and intestinal fluids at a concentration of 1 mg / mL. After incubation at 200 rpm and 37 °C for 2 h, the morphology and structure of the kudzu root vesicle-like nanoparticles under different treatments were observed by transmission electron microscopy.

[0063] Depend on Figure 5 It can be seen that the morphology of kudzu vesicle-like nanoparticles did not change before and after digestion, and their structure remained intact. This indicates that kudzu vesicle-like nanoparticles have the ability to resist gastrointestinal digestion and can pass through the gastrointestinal tract to reach inflamed intestinal sites via oral delivery.

[0064] 5. Lipidome analysis of kudzu vesicle-like nanoparticles

[0065] Lipids were extracted from kudzu vesicle-like nanoparticles using organic reagents, and the lipids were sequenced and analyzed using a high-performance liquid chromatography-mass spectrometry system.

[0066] Depend on Figure 6 As can be seen from A, the lipids in the vesicle-like nanoparticles of kudzu root are mainly glycerol lipids, glycerophospholipids, and sphingolipids. The lipid subclasses mainly include triglycerides, diglycerides, ceramides, phosphatidylcholine, hexosylceramide, and phosphatidylethanolamine. Figure 6 KEGG analysis of B showed that lipids in kudzu vesicle-like nanoparticles are mainly related to various cellular processes (transport and catabolism, cell growth and death), environmental information processing (signal molecules and interactions, signal transduction), metabolism (lipid metabolism, biosynthesis and metabolism of glycans, carbohydrate metabolism, amino acid metabolism, etc.), and various organic systems (sensory system, nervous system, immune system, endocrine system, etc.).

[0067] 6. Quantitative analysis of puerarin content by high performance liquid chromatography

[0068] Accurately weigh 5 mg of puerarin standard, dissolve in 5 mL of methanol (HPLC grade) to make a 1 mg / mL stock solution. Take 400 μL of the stock solution, dilute to 2 mL, vortex mix, then take 1 mL again, dilute to 2 mL, repeat this step, and continuously dilute the standard to prepare the standard curve. (Standard curve concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.125 μg / mL). Mix according to the ratio of puerarin vesicle-like nanoparticles: methanol = 1:9, vortex for 30 s, centrifuge at 12000 g for 10 min, take the supernatant for high performance liquid chromatography analysis. Use a C18 reverse phase chromatographic column (specifications: 4.6 x 250 mm, filler particle size: 5 μm) produced by Agilent Company for separation. The mobile phase system is composed of acetonitrile and 0.1% phosphoric acid aqueous solution, with a volume ratio of 12:88. The chromatographic conditions are set as follows: constant flow rate 1.0 mL / min, UV detection wavelength 250 nm, column temperature control at 30°C. Each injection is 10 μL. Obtain the peak area signal of the target compound in each sample by high performance liquid chromatography. Use the external standard method to quantify and establish a standard curve with a series of gradient concentrations of puerarin standard solution, where the standard concentration (μg / mL) is the abscissa (X axis), and the corresponding chromatographic peak area is the ordinate (Y axis). According to the standard curve equation, combined with the chromatographic peak area value of puerarin in the puerarin vesicle-like nanoparticle sample, calculate its content.

[0069] Figure 7 A is the established standard curve, which is Figure 7 B, it can be known that the content of puerarin in puerarin vesicle-like nanoparticles is 35.4 ± 1.2 μg / mg of puerarin vesicle-like nanoparticles.

[0070] Example 2: Therapeutic application of puerarin vesicle-like nanoparticles in ulcerative colitis

[0071] I. Establishment of animal model of ulcerative colitis and administration of puerarin vesicle-like nanoparticles for treatment

[0072] An ulcerative colitis animal model was established using dextran sodium sulfate (DSS). After 7 days of acclimation, 15 C57BL / 6 male mice were randomly divided into 3 groups, 5 mice in each group: healthy control group, DSS control group, DSS + pueraria vesicle-like nanoparticle group. Except for the healthy control group, the remaining two groups of mice were given 2% DSS (w / v) in the drinking water for 7 consecutive days, with fresh DSS solution replaced every other day. At the same time of drinking DSS, except for the healthy control group of mice, the DSS group of mice were given 200 μL of phosphate buffer by gavage daily, and the DSS + pueraria vesicle-like nanoparticle group of mice were given 0.35 mg of pueraria vesicle-like nanoparticles by gavage daily. The body weight change, fecal consistency and bleeding of the mice were monitored daily, and the disease activity index (disease activity index = body weight loss score (<1% - 0, 1-5% - 1, 6-10% - 2, 11-20% - 3, >20% - 4) + fecal state score (normal - 0, soft stool - 1, loose stool - 2, watery stool - 3) + bleeding score (normal - 0, mild bleeding - 1, obvious bleeding - 2, massive bleeding - 3)) was used for scoring. The mice were sacrificed on the 7th day, and the colon of the mice was taken, photographed, and the corresponding tissues were saved.

[0073] As can be seen from Figure 8 and Figure 9 , the body weight of the healthy control group of mice increased steadily, the fecal condition was normal, and there was no bleeding phenomenon. From the 3rd day, the body weight of the DSS group of mice began to decrease, and then continuously fell to about 90%, accompanied by diarrhea and fecal bleeding, and the disease activity index increased significantly. After treatment with pueraria vesicle-like nanoparticles, the phenomenon was significantly alleviated, the body weight of the mice decreased slowly, and was about 97% of the initial body weight, and the disease activity index also decreased, indicating that pueraria vesicle-like nanoparticles significantly alleviated the symptoms of colitis.

[0074] As can be seen from Figure 10 , the colon length of the healthy control group of mice was 7.29 cm, the colon of the DSS group was significantly shortened, only 4.79 cm, and after administration of pueraria vesicle-like nanoparticles, the colon length increased to 5.83 cm. This also effectively indicates that pueraria vesicle-like nanoparticles can alleviate ulcerative colitis.

[0075] In previous studies, puerarin was typically used at doses of 50-200 mg / kg to treat colitis. Subsequent studies used puerarin vesicular nanoparticles at a dose of only 15 mg / kg, indicating that puerarin vesicular nanoparticles can achieve the same effect as high-dose puerarin at a lower dose. Furthermore, the puerarin content in the 15 mg / kg puerarin vesicular nanoparticles was only 0.531 mg / kg, far below the dose required for puerarin to exert its effect. This result suggests that the effect of puerarin vesicular nanoparticles in alleviating IBD may be due to other active substances, or it may be due to the excellent delivery effect of the puerarin vesicular nanoparticles, allowing low-dose puerarin to achieve the same effect as high-dose puerarin.

[0076] II. Assessment of colonic pathological damage in mice using hematoxylin and eosin (H&E) staining

[0077] Distal colon tissue was taken and fixed by immersion in 4% paraformaldehyde for 24 hours. After rinsing with running water for 4-6 hours to remove excess paraformaldehyde, H&E staining analysis was then performed.

[0078] Depend on Figure 11 It was found that the DSS group mice exhibited significant inflammatory cell infiltration in the intestines, disruption of the intestinal epithelial barrier, disappearance of crypt structures, and a reduction in goblet cells. In contrast, the intestines of mice orally administered kudzu vesicle-like nanoparticles were similar to those of the healthy control group, with intact crypt structures, a significant increase in goblet cells, and no significant inflammatory cell infiltration, indicating that kudzu vesicle-like nanoparticles effectively alleviated colitis in mice.

[0079] This invention provides a method for extracting vesicular nanoparticles from kudzu root. These vesicular nanoparticles are approximately 200 nm in size and have a negative zeta potential. They exhibit good gastrointestinal stability, maintaining their morphology and structure intact after digestion in simulated gastrointestinal fluids. They can reach and remain at the site of inflammation, exerting their therapeutic effect. The vesicular nanoparticles are rich in lipids and proteins, playing important roles in biological processes such as metabolism, various cellular processes, and carbohydrate and amino acid metabolism. They also contain trace amounts of puerarin. This invention is the first to extract stable and homogeneous vesicular nanoparticles from kudzu root decoction after high-temperature boiling. This provides a new approach for exploring novel active ingredients in traditional Chinese medicine and a new method for treating ulcerative colitis.

[0080] In summary, the application provides a extraction method of Pueraria lobata vesicle-like nanoparticles and application thereof, the Pueraria lobata vesicle-like nanoparticles as a new active ingredient in Pueraria lobata are nanostructures carrying proteins, lipids and other active substances, which improve the bioavailability of effective components in Pueraria lobata, can treat ulcerative colitis by relieving intestinal inflammation, restoring intestinal barrier and balancing intestinal microecology; and the Pueraria lobata vesicle-like nanoparticles have good gastrointestinal stability and inflammation site retention, so that they can be used as oral preparations, greatly improving patient compliance. Pueraria lobata is widely available, and the vesicle-like nanoparticle extraction process is simple, so that the Pueraria lobata vesicle-like nanoparticles have the characteristics of easy acquisition and low cost, and have superior biocompatibility, no obvious toxicity to healthy tissue cells, low immunogenicity, small risk, and are suitable for commercial production. The application extracts stable vesicle-like nanoparticles from Pueraria lobata decoction liquid subjected to high-temperature decoction, which also provides a reference for subsequent research on vesicle-like nanoparticles in dried plants; provides a new idea for exploring active substances for treating ulcerative colitis in traditional Chinese medicine, promotes the integration of traditional Chinese medicine and modern medicine; and provides a new route for the use of traditional Chinese medicinal materials.

[0081] It should be noted that in this document, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusions, so that processes, methods, articles or devices including a series of elements not only include those elements, but also include other elements not explicitly listed, or include elements inherent to such processes, methods, articles or devices. Without more limitations, the element defined by the statement "including a" does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0082] The above-mentioned application example serial numbers are only for description, not representing the advantages and disadvantages of the embodiments.

[0083] The above embodiments are only used to illustrate the technical solutions of the application and not limit the application, although the application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the application can be modified or replaced equivalently without departing from the purpose and scope of the application, and all should be covered in the scope of the claims of the application.

Claims

1. A method of extracting Pueraria montana var. lobata Radix vesicle-like nanoparticles, characterized by, The method comprises the following steps: S1, crushing dry kudzu root powder, adding ultrapure water to decoct for 30-60 min, cooling, and then squeezing and filtering through gauze to obtain kudzu root decoction; S2, taking the kudzu root decoction of S1 and sequentially performing gradient centrifugation at 4-8℃ for 10-50 min at 1000-12000g, taking the supernatant to obtain a crude extract of kudzu root vesicle-like nanoparticles; S3, taking the crude extract of S2 and performing ultracentrifugation at 4-8℃ at 150000-170000g, discarding the supernatant, resuspending the bottom precipitate with phosphate, and obtaining a crude extract of kudzu root vesicle-like nanoparticles; S4, taking the obtained crude extract and performing low-speed centrifugation at 4-8℃ at 4000-6000rpm, taking the supernatant, and filtering through a filter membrane to obtain kudzu root vesicle-like nanoparticles.

2. The method of claim 1, wherein, In step S1, The crushing time is 20-40s; When adding ultrapure water for decoction, the w / v of dry kudzu root powder and ultrapure water is 1:6-1:10, the decoction time is 45 min, and the gauze aperture is 100 mesh.

3. The method of claim 1, wherein, In step S2, The gradient centrifugation is specifically 1000-1500g for 10-15 min, 3000-5000g for 20-30 min, and 10000-12000g for 30-50 min.

4. The method of claim 1, wherein, In step S3, the ultracentrifugation is 150000g for 70-90 min.

5. The method of claim 1, wherein, In step S4, the low-speed centrifugation is 5000rpm for 2-5 min, and the filter membrane aperture is 0.22μm.

6. Kudzu root vesicle-like nanoparticles prepared by the method of any one of claims 1-5.

7. The fenugreek vesicular nanoparticle of claim 6, wherein, The particle size of the kudzu root vesicle-like nanoparticles is 100-300nm, and the potential of the kudzu root vesicle-like nanoparticles is -3--9mv.

8. Use of the kudzu root vesicle-like nanoparticles of claim 6 or 7 in the preparation of a medicament for treating colitis.

9. Use according to claim 8, characterized in that, The colitis is ulcerative colitis.

10. A pharmaceutical preparation, characterized in that, The kudzu root vesicle-like nanoparticles of claim 6 or 7 and a pharmaceutically acceptable carrier.

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