Fuzhuan tea vesicle-like nanoparticles as well as decoction preparation method and application thereof

Abstract

The invention relates to a decoction preparation method of Fuzhuan tea vesicle-like nanoparticles. The decoction preparation method comprises a Fuzhuan tea stock solution preparation step, a Fuzhuan tea vesicle-like nanoparticle preparation step and a Fuzhuan tea vesicle-like nanoparticle purification preparation step. Meanwhile, the invention further provides the Fuzhuan tea vesicle-like nano-particles prepared by the method and application of the Fuzhuan tea vesicle-like nano-particles. The tea raw material source is not influenced by seasons, organic reagents are completely not used, the damage of environmental pollution and organic reagent residues to human bodies is avoided, the preparation cost is greatly reduced, and the Fuzhuan tea vesicle-like nanoparticles have the characteristics of gastrointestinal digestion resistance, good intestinal distribution and good safety, and can be used for preparing the Fuzhuan tea vesicle-like nanoparticles. It is proved that the decocted Fuzhuan tea vesicle-like nanoparticles can better improve the symptoms of colitis and can be applied to oral administration for treating gastrointestinal diseases, and meanwhile the yield of the Fuzhuan tea vesicle-like nanoparticles is increased.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to tea vesicle-like nanoparticles and a preparation method and application thereof. Background Art

[0002] Fucha, one of the treasures of ancient China's Silk Road, is a fermented black tea that has been brewed from boiling water and drunk for centuries.

[0003] Extracellular vesicles (EVs) are lipid bilayer vesicles secreted into the extracellular space by all cell types. EVs are roughly divided into three main types, including exosomes (30-150nm), microvesicles (150-1000nm), and apoptotic bodies (1-5μm).

[0004] Ulcerative colitis is an idiopathic chronic inflammatory disease with a fairly high incidence worldwide. Current therapeutic drugs, such as 5-aminosalicylate (5-ASA), immunosuppressants (such as methotrexate), and biologics (such as infliximab, TNF-α), mainly target single-factor treatments for IBD, but their clinical efficacy is not ideal. In addition, these drugs often lack target specificity, have insufficient residence time in the intestine, and are accompanied by systemic adverse reactions, resulting in a low remission rate for patients. Therefore, a synergistic intervention strategy is needed to restore intestinal metabolic balance.

[0005] Oral administration is the preferred route of administration, but the challenges of oral administration are huge. Due to the harsh gastrointestinal environment, it is difficult for drugs to be completely absorbed by the human body through the gastrointestinal wall. For many years, people have been exploring the application of functionalized nano drug delivery systems to improve the bioavailability of drugs after oral administration, thereby improving the poor targeting of current drugs for the treatment of ulcerative colitis and reducing side effects.

[0006] Existing studies have shown that mammalian exosomes can be used to treat ulcerative colitis, but their low yield and difficulty in large-scale production limit their application. Recent studies have found that plant-derived vesicle-like nanoparticles exist in plants, which are a type of cutting-edge, harmless, environmentally friendly, and powerful nanocarriers that are less immunogenic and harmful than mammalian exosome particles.

[0007] For example, the Chinese patent application "Therapeutic Use of Bidens pilosa Extracellular Vesicles for Inflammatory Bowel Disease" with application publication number CN117982551A adopts a squeezing method to extract fresh plant vesicle-like nanoparticles for oral use in inflammatory bowel disease. However, the Bidens pilosa used has high freshness requirements, and the preparation method mainly adopts squeezing, which will inevitably have a great impact on the cost and purity of the vesicle-like nanoparticles.

[0008] The currently established methods for preparing tea-based vesicle-like nanoparticles include the preparation method of ultracentrifugation after squeezing. The Chinese patent application "A Fusion Nanovesicle and Its Preparation Method and Application" with application publication number CN117100700A uses squeezing method and sucrose gradient purification to extract tea vesicle-like nanoparticles for the construction of fusion vesicle-like nanoparticles, and its preparation must use fresh tea leaves.

[0009] The existing preparation methods of Fucha extracts mostly rely on a large number of organic reagents and are cumbersome, which are prone to pollution and organic reagent residues, causing potential damage to the human body. The Chinese patent application "A kind of Hucha granules for regulating gastrointestinal function" with application publication number CN107467277A, "a kind of Hucha granules for regulating gastrointestinal function", the preparation method of the Hucha granules for regulating gastrointestinal function is to add Hucha to acidic water, boil at 100°C for 30 minutes, filter with filter cloth, combine the two filtrates, concentrate under reduced pressure, decolorize with activated carbon, centrifuge, and vacuum dry. The preparation method relies on a large number of organic reagents and is cumbersome, and there is no way to overcome the problems of organic reagent residues, environmental pollution, and human body damage, and the absorption has the characteristics of poor targeting.

[0010] It can be seen that the existing technical solutions have high requirements for the freshness of the raw materials used in the preparation of vesicle-like nanoparticles, and the preparation method mainly adopts squeezing, which makes the cost and purity of vesicle-like nanoparticles fluctuate greatly due to seasonality, and it is not easy to control the quality of raw materials. More importantly, there is no relevant research on how to increase the number of vesicle-like nanoparticles;

[0011] At the same time, the extraction of existing Pu'er tea products mostly relies on organic reagents, and it is difficult to overcome the problems of organic reagent residues and pollution. Its application in intestinal treatment is only mentioned, such as irritable bowel syndrome. The existing description of tea vesicle-like nanoparticles can only prove that tea vesicle-like nanoparticles have anti-inflammatory, antioxidant and anti-tumor effects, and no further research has been done on ulcerative colitis. More importantly, the prepared extracts have poor targeting and obvious side effects during oral application.

[0012] Therefore, it is urgently needed to develop a Pu'er tea vesicle-like nanoparticle that does not rely on organic reagents throughout the process, is green, environmentally friendly, and has controllable quality, can overcome the digestion of strong acids and digestive enzymes when taken orally, and thus exert a therapeutic effect on intestinal diseases through oral administration. Summary of the invention

[0013] The purpose of the present invention is to avoid the shortcomings of the prior art and provide a method for preparing and applying vesicle-like nanoparticles of Fu tea, which can be obtained from processed Fu tea without organic reagents and can be used to treat ulcerative colitis.

[0014] To achieve the above object, the technical solution adopted by the present invention is: a decoction preparation method of Fucha vesicle-like nanoparticles, comprising the following steps:

[0015] Step 1: Preparation of Fucha stock solution:

[0016] The puer tea is crushed until at least 60-80% of the tea powder passes through a 100-200 mesh sieve, the sieved tea powder is brewed with water, the mass ratio of the tea powder to water is 1:6-1:10, and after boiling for at least 30 minutes, it is squeezed through a 100-200 mesh filter to obtain a puer tea stock solution;

[0017] Step 2: Preparation of Fucha vesicle-like nanoparticle solution:

[0018] The supernatant obtained by gradient centrifugation of the Fu Tea stock solution is placed in a container, and then centrifuged at a relative centrifugal force higher than the highest relative centrifugal force of the gradient centrifugation, and then a standard phosphate buffer with a mass ratio of 1:5 to 1:10 is added to resuspend the precipitate, so as to obtain Fu Tea vesicle-like nanoparticles;

[0019] Step 3: Purification and preparation of Fucha vesicle-like nanoparticle solution:

[0020] Prepare sucrose solutions with concentrations of 8%, 30%, 45%, and 60%, respectively, add the sucrose solutions into a container in order from low concentration to high concentration, then add the Fucha vesicle-like nanoparticle solution diluted 8 to 10 times with a standard phosphate buffer, wherein the volume ratio of the sucrose solution to the Fucha vesicle-like nanoparticle solution is 15:38.5 to 18:38.5, and after the formed mixed solution is centrifuged to obtain a strip containing Fucha vesicle-like nanoparticles, and suck out the strip distributed in the sucrose density layer between 8% and 30% in the container;

[0021] The strip was diluted with standard phosphate buffer at a ratio of 1:6 to 1:10, and the residual sucrose solution in the strip was removed by centrifugation. The supernatant was then removed, and the precipitate in the container was diluted with standard phosphate buffer at a ratio of 1:1 to 1:5 to obtain purified Fucha vesicle-like nanoparticles.

[0022] Furthermore, the Fu tea in step one is a washed Fu tea, and the washing process is: weigh the Fu tea and pour it into a container, wash it with pure water at least 3 times, then wash it with distilled water at least 3 times, and then add 6 to 10 times of distilled water to soak for at least 30 minutes to ensure that the leaves are completely wet.

[0023] Furthermore, the gradient centrifugation described in step 2 is specifically: the Pu'er tea stock solution is centrifuged at a relative centrifugal force of 500g-1500g for 10-20min, at a relative centrifugal force of 2000g-4000g for 20-30min, and at a relative centrifugal force of 8000g-13000g for 40-60min to remove large particles and crude fiber in the Pu'er tea.

[0024] Furthermore, the relative centrifugal force of 100000-150000 g, which is higher than the highest relative centrifugal force of the gradient centrifugation in step 2, and the centrifugation time is 60-90 min, are used to enrich the Fu'er tea vesicle-like nanoparticles.

[0025] Furthermore, the mixed solution in step 3 is centrifuged at a relative centrifugal force of 100,000 to 150,000 g for a centrifugal time of 90 to 120 min;

[0026] The relative centrifugal force of the centrifugal treatment to remove the residual sucrose solution in the strip is 100,000 to 150,000 g, and the centrifugal time is 60 to 90 minutes.

[0027] Furthermore, the Fu tea is Fu tea or dark tea made from dark tea.

[0028] The present invention also provides a kind of Fucha vesicle-like nanoparticles obtained by the preparation method, wherein the Fucha vesicle-like nanoparticles extracted by decoction have a particle size of 300-480 nm and a yield of 2×10 10 The number of particles per gram of Fu tea appears disc-shaped or tray-shaped under a transmission electron microscope and a lipid membrane structure under a scanning electron microscope.

[0029] The present invention also provides an application of the Fucha vesicle-like nanoparticles in a drug for treating ulcerative colitis.

[0030] Furthermore, it also includes oral application in drugs for treating ulcerative colitis. The Efu tea vesicle-like nanoparticles can overcome the strong acid environment of gastric juice, the alkaline environment of intestinal juice and the digestive effect of the gastrointestinal tract during oral administration, and can stably exist in the human stomach and intestines.

[0031] The beneficial effects of the present invention are:

[0032] (1) Extract vesicle-like nanoparticles from Fucha by squeezing, purify and characterize them;

[0033] (2) The first method to extract nanoparticles from Fucha vesicles after decoction and its characterization:

[0034] (3) For the first time, the stability of the decocted Fu'an tea vesicle-like nanoparticles and the comparative squeezed Fu'an tea vesicle-like nanoparticles in simulated gastrointestinal fluid was systematically compared;

[0035] (4) The in vivo distribution of oral comparative samples of squeezed and boiled Fu'er tea vesicle-like nanoparticles was reported for the first time;

[0036] Compared with the prior art which focuses on vesicle-like nanoparticles derived from fresh plants, the source of tea raw materials used in the present invention is not affected by seasons, which greatly reduces the preparation cost. Compared with the existing reported extraction methods, the Fu'an tea vesicle-like nanoparticles are resistant to gastrointestinal digestion and have good distribution in the gastrointestinal tract. This proves that decocted Fu'an tea vesicle-like nanoparticles can better improve the symptoms of colitis and can be used for oral administration to treat gastrointestinal diseases. At the same time, the output of Fu'an tea vesicle nanoparticles is also increased.

[0037] The extraction method provided by the present invention greatly improves the symptoms of ulcerative colitis and has great potential in the treatment of ulcerative colitis. It has high bioavailability for oral application, tolerates gastrointestinal digestion, and does not rely on organic reagents throughout the process, thus overcoming the bottleneck of the prior art and providing a new treatment strategy for the current treatment of inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the particle number detection result of the vesicle-like nanoparticles of Fu tea squeezed in the comparative example;

[0039] Figure 2 The transmission electron microscopy image of the vesicle-like nanoparticles of Fu tea squeezed from the comparative example;

[0040] Figure 3 This is a scanning electron microscope image of the vesicle-like nanoparticles of Fu tea squeezed in the comparative example;

[0041] Figure 4 This is a graph showing the result of detecting the number of particles of the vesicle-like nanoparticles of the decoction of Fu tea of ​​the present invention;

[0042] Figure 5 This is a transmission electron microscope image of the vesicle-like nanoparticles of the decoction of Fu tea of ​​the present invention;

[0043] Figure 6 This is a scanning electron microscope image of the vesicle-like nanoparticles of the decoction of Fu tea of ​​the present invention;

[0044] Figure 7 This is a yield evaluation diagram of the comparative example of squeezing the vesicle-like nanoparticles of Fu'er tea and the decocted vesicle-like nanoparticles of Fu'er tea of ​​the present invention, wherein * refers to p < 0.05, ** refers to p < 0.01, *** refers to p < 0.001, **** refers to p < 0.0001, and ns refers to no significant difference;

[0045] Figure 8 This is a comparative diagram of the evaluation of the digestion resistance of the Fucha vesicle-like nanoparticles extracted from the comparative example;

[0046] Fig. 9 This is a comparative diagram of the evaluation of the anti-digestion ability of the vesicle-like nanoparticles of boiled Fu'er tea of ​​the present invention;

[0047] Fig.10 This is the gastrointestinal distribution diagram of the Fucha vesicle-like nanoparticles extracted from the comparative example;

[0048] Fig.11 This is a gastrointestinal distribution diagram of the vesicle-like nanoparticles of boiled Fu tea of ​​the present invention;

[0049] Fig.12 It is a schematic diagram of the comparative example of squeezing the Fu tea vesicle-like nanoparticles and the decocted Fu tea vesicle-like nanoparticles provided by the present invention for the treatment of ulcerative colitis.

[0050] Fig.13 This is a photo of the colon after using the comparative example of squeezed Fu tea vesicle-like nanoparticles and the decocted Fu tea vesicle-like nanoparticles provided by the present invention to treat ulcerative colitis.

[0051] Fig.14 This is an evaluation chart of the disease activity index after the comparative example of squeezed Fu tea vesicle-like nanoparticles and the decocted Fu tea vesicle-like nanoparticles provided by the present invention were used to treat ulcerative colitis, wherein * refers to p < 0.05, ** refers to p < 0.01, *** refers to p < 0.001, **** refers to p < 0.0001, and ns refers to no significant difference.

[0052] Fig.15 This is an evaluation graph of the colon length after the comparative example of squeezed Fu tea vesicle-like nanoparticles and the decocted Fu tea vesicle-like nanoparticles provided by the present invention were used to treat ulcerative colitis, wherein * refers to p < 0.05, ** refers to p < 0.01, *** refers to p < 0.001, **** refers to p < 0.0001, and ns refers to no significant difference.

[0053] Fig.16 This is a comparative diagram of hematoxylin-eosin pathological staining of the colon after the comparative example of squeezing Fu'er tea vesicle-like nanoparticles and the decocted Fu'er tea vesicle-like nanoparticles provided by the present invention were used to treat ulcerative colitis;

[0054] Fig.17 This is a strip distribution diagram of the Fucha vesicle-like nanoparticles of the present invention;

[0055] Fig.18 It is a safety assessment diagram of the body during oral administration of the comparative example of squeezed Fu tea vesicle nanoparticles and the decocted Fu tea vesicle nanoparticles of the present invention. DETAILED DESCRIPTION

[0056] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0057] In order to achieve the above object, the present invention provides the following specific implementation methods:

[0058] Example 1: Fig.17 As shown, a method for preparing pueraria tea vesicle-like nanoparticles by decoction comprises the following steps:

[0059] Fucha is made from black tea or black tea

[0060] Step 1: Preparation of Fucha stock solution:

[0061] Weigh the Pu'er tea and pour it into a container. Wash it at least 3 times with purified water and then at least 3 times with distilled water. Then add 6 to 10 times the amount of distilled water and soak it for at least 30 minutes to ensure that the leaves are completely moistened.

[0062] The puer tea is crushed until at least 60-80% of the tea powder passes through a 100-200 mesh sieve, the sieved tea powder is brewed with water, the mass ratio of the tea powder to water is 1:6-1:10, and after boiling for at least 30 minutes, it is squeezed through a 100-200 mesh filter to obtain a puer tea stock solution;

[0063] Step 2: Preparation of Fucha vesicle-like nanoparticle solution:

[0064] The Fu tea stock solution is centrifuged at a relative centrifugal force of 500g-1500g for 10-20min, at a relative centrifugal force of 2000g-4000g for 20-30min, and at a relative centrifugal force of 8000g-13000g for 40-60min to remove large particles and crude fibers in the Fu tea, and the obtained supernatant is placed in a container, and then centrifuged at a relative centrifugal force of 100000-150000g for 60-90min, and then a standard phosphate buffer with a mass ratio of 1:5-1:10 is added to resuspend the precipitate, so as to obtain Fu tea vesicle-like nanoparticles;

[0065] Step 3: Purification and preparation of Fucha vesicle-like nanoparticle solution:

[0066] Sucrose solutions with concentrations of 8%, 30%, 45%, and 60% are prepared, and the sucrose solutions are added into a container in order from low concentration to high concentration, and then added into the Fucha vesicle-like nanoparticle solution diluted 8 to 10 times with a standard phosphate buffer, the volume ratio of the sucrose solution to the Fucha vesicle-like nanoparticle solution is 15 to 18:38.5, and the formed mixed solution is subjected to centrifugation at a relative centrifugal force of 100000 to 150000 g and a centrifugation time of 90 to 120 min to obtain a strip containing Fucha vesicle-like nanoparticles, and the strip distributed between 8% and 30% of the sucrose density layer in the container is sucked out, such as Fig.17 As shown;

[0067] The strip is diluted with standard phosphate buffer at a dilution ratio of 1:6 to 1:10, and the strip is centrifuged at a relative centrifugal force of 100,000 to 150,000 g for 60 to 90 min to remove the residual sucrose solution in the strip. The supernatant is then discarded, and the precipitate in the container is diluted with standard phosphate buffer at a dilution ratio of 1:1 to 1:5 to obtain purified Fucha vesicle-like nanoparticles.

[0068] Example 2: Figure 1-Figure 7 As shown, the present invention also provides a kind of Fucha vesicle-like nanoparticles obtained by the preparation method, wherein the Fucha vesicle-like nanoparticles extracted by decoction have a particle size of 300-480 nm and a yield of 2×10 10 The number of particles per gram of Fu tea appears disc-shaped or tray-shaped under a transmission electron microscope and a lipid membrane structure under a scanning electron microscope.

[0069] In order to further illustrate the technical scheme and effect of the present invention, the following test examples and comparative examples are provided, wherein the comparative example is to obtain a Fucha vesicle-like nanoparticle solution by squeezing, specifically:

[0070] ① Crude extraction of Fucha vesicle-like nanoparticles:

[0071] Take 100g of Fu tea and divide it into two portions of 50g each;

[0072] Comparative Example Extraction Method: After washing, pour one portion into a juicer, then add 300 mL of ultrapure water into the juicer, extract the juice for 2 minutes, and then squeeze it with a 100-200 mesh filter to obtain the extracted Fu tea stock solution;

[0073] The decoction method of the present invention comprises the following steps: another portion is crushed by a grinder, the tea powder is passed through a 100-mesh screen, and the screened tea powder is brewed with water, wherein the mass ratio of the tea powder to water is 1:6, and after decocting for 30 minutes, the decoction is passed through a 200-mesh screen and squeezed to obtain the decoction Fu tea stock solution.

[0074] Pour the two portions of Fucha stock solution into two centrifuge bottles and centrifuge them at 1000g for 10 min, 3000g for 20 min, and 10000g for 40 min. After the gradient centrifugation, pour the supernatant into the ultracentrifuge tube and balance it. Centrifuge it at 150000g for 70 min. After the centrifugation, add 5 mL of phosphate buffer to resuspend the precipitate, and collect it for subsequent sucrose purification.

[0075] ② Purify the Fucha vesicle-like nanoparticles of the comparative example and the test example of the present invention respectively:

[0076] Prepare sucrose solutions with concentrations of 8%, 30%, 45%, and 60%, and slowly add the sucrose solutions into the ultracentrifuge tube from low concentration to high concentration, then add the crude extracted Fu'an tea vesicle-like nanoparticles solution diluted with standard phosphate solution, balance the centrifuge tube and centrifuge at 150,000 g for 90 minutes, you can see the bands containing Fu'an tea vesicle-like nanoparticles, Fu'an tea vesicle-like nanoparticles are mainly distributed between the 8% and 30% bands, suck out the bands with a pipette, and put them into a 15 mL centrifuge tube for subsequent desugaring.

[0077] The purified exosome strip solution was added to a new ultracentrifuge tube and centrifuged at 150,000 g for 70 min to remove the residual sucrose solution in the strip, the supernatant was discarded, and the precipitate was resuspended with standard phosphate buffer to obtain the purified comparative example squeezed Fu tea vesicle-like nanoparticles and the decoction Fu tea vesicle-like nanoparticles of the present invention, which were then filtered and collected using a 0.22 μm filter membrane for subsequent characterization.

[0078] ③ Take the above products respectively, dilute them 1:6, and then characterize them.

[0079] 1) Nanoparticle Tracking Analysis (NTA) characterization of particle size and particle count: The specific steps are:

[0080] ① Clean the sample pool with deionized water; the instrument is calibrated with polystyrene microspheres;

[0081] ② Wash the sample pool with 1X PBS;

[0082] ③The sample was diluted with 1X PBS and the particle size and particle number were measured.

[0083] Comparative proportion extraction method: Figure 1 As shown, Figure 1 The particle size distribution of the vesicle-like nanoparticles extracted from the comparative example of Fucha is mostly around 200 nm;

[0084] Test example of the present invention decoction method: Figure 4 As shown, Figure 4It is shown that the particle size distribution of the vesicle-like nanoparticles of decoction of Fu tea is mostly around 220nm; Figure 5 It was shown that the vesicle-like nanoparticles of boiled Fu tea also appeared disc-shaped or tray-shaped under transmission electron microscopy.

[0085] The yield is measured by the ratio of the number of particles to the mass of Fu'er tea, and the yield of the comparative example squeezing method and the yield of the inventive example decoction method can be obtained respectively.

[0086] Comparative Example Extraction Method Output: Figure 7 As shown, the yield of the Fucha vesicle-like nanoparticles extracted from the comparative example provided by the comparative example is 5×10 9 Number of particles / g of Fu tea.

[0087] Invention Example Decoction Method Yield: Figure 7 The yield of the Fucha vesicle-like nanoparticles extracted by decoction provided by the present invention is 2×10 10 The number of particles / gram of Fu'er tea can be compared with the yield of the comparative example and the inventive example to show that with the same mass of Fu'er tea, the yield of the Fu'er tea vesicle-like nanoparticles extracted by decoction of the present invention is 4 times that of the Fu'er tea vesicle-like nanoparticles extracted by the comparative example.

[0088] 2) Transmission electron microscopy characterization:

[0089] Take 30 μL of the mixed sample and drop it on the copper mesh of the carbon support film, let it stand for 5 minutes; use filter paper to absorb the residual liquid from the edge of the copper mesh. Add 2% phosphotungstic acid to the copper mesh, dye it at room temperature for 30 seconds, and use filter paper to absorb the excess dye; dry it naturally. Observe under JEM-1400FLASH transmission electron microscope.

[0090] Comparative proportion extraction method: Figure 2 As shown, Figure 2 The vesicle-like nanoparticles of Fu tea squeezed in the comparative example appeared in the shape of a saucer or a tea tray under a transmission electron microscope;

[0091] Test example of the present invention decoction method: Figure 5 As shown in the figure, the vesicle-like nanoparticles of the decoction of Fu tea of ​​the present invention also appear in the shape of a disc or a tea tray under a transmission electron microscope.

[0092] 3) Scanning electron microscopy characterization:

[0093] Take 10 μL of the mixed sample and drop it on the silicon wafer, air-dry it naturally, and observe it under a JSM-7610FPLΜS scanning electron microscope.

[0094] Comparative proportion extraction method: Figure 3 As shown, Figure 3 The lipid membrane structure of the comparative example extracted Pu'er tea vesicle-like nanoparticles under scanning electron microscopy is shown in FIG.

[0095] Test example of the present invention decoction method: Figure 6 As shown, Figure 6 It is shown in the figure that the vesicle-like nanoparticles of the decoction of Fu tea of ​​the present invention present a lipid membrane structure under a scanning electron microscope.

[0096] Example 3: Figure 8-Figure 16 As shown, the present invention also provides an application of Fucha vesicle-like nanoparticles in a drug for treating ulcerative colitis and an oral application of the drug.

[0097] In order to further illustrate the technical application effect of the present invention, the following experimental evidence is provided, in which the comparative test results of the extraction of Fucha are compared with the effect of the present invention:

[0098] 1) Preparation of simulated gastric and intestinal fluids:

[0099] Preparation of simulated gastric fluid: 80 mM hydrochloric acid, 35 mM sodium chloride, 0.3% pepsin, add deionized water to make up to 100 mL, pH 1.2.

[0100] Preparation of simulated intestinal fluid: 15 mM sodium hydroxide, 50 mM potassium dihydrogen phosphate, 1% pancreatic enzyme, add deionized water to make up to 100 mL, pH 6.8.

[0101] 2) The comparative example squeezed Pu'er tea vesicle-like nanoparticles and the decocted Pu'er tea vesicle-like nanoparticles of the present invention were diluted to a concentration of 0.7 mg / mL with standard phosphate buffer, simulated gastric juice, and intestinal juice, respectively, and incubated on a shaker at 37°C and 200 rpm for 2 h. The changes in the number of Pu'er tea vesicle-like nanoparticles in the simulated gastric juice and simulated intestinal juice were then determined by NTA.

[0102] 3) Prepare the fluorescent dye DiR solution. DiR is a lipophilic fluorescent dye that is often used to stain cell membranes and other fat-soluble biological structures. Since DiR's near-infrared fluorescence can penetrate cells and tissues, it is also often used in small animal in vivo imaging technology. To show the in vivo distribution of Fucha vesicle-like nanoparticles. Weigh 2 mg DiR, add 197 μL dimethyl bissulfone to dissolve, prepare a stock solution with a final concentration of 10 mM, dilute with sodium chloride to 10 μM, and heat at 37°C until fully dissolved to form a working solution;

[0103] 4) combining the prepared DiR with the comparative example squeezed Fu tea vesicle-like nanoparticles and the decoction Fu tea vesicle-like nanoparticles of the present invention, so that the concentrations of the comparative example squeezed Fu tea vesicle-like nanoparticles and the decoction Fu tea vesicle-like nanoparticles of the present invention are both 1 mg / mL;

[0104] 5) Eight-week-old C57BL6J mice were depilated with a depilatory cream, and then gavage was performed with 0.2 mL of the DiR-bound control sample squeezed Fu'er tea vesicle-like nanoparticles and 0.2 mL of the DiR-bound decocted Fu'er tea vesicle-like nanoparticles of the present invention, respectively. After 6 hours, the mice were dissected and the fluorescence intensity of the gastrointestinal tract was observed using a live imaging instrument.

[0105] 6) If Figure 7-16 As shown, the model of inflammatory bowel disease was established:

[0106] Twenty 7-week-old male C57BL / 6J mice were raised in the Experimental Animal Center of the Medical Research Institute of Northwestern Polytechnical University. After the mice were fed with a free diet for one week, five mice were randomly selected as a normal group, and the other experimental mice were randomly divided into three groups: a DSS modeling group, a control group of extracted Fu'an tea vesicle-like nanoparticles, and a group of boiled Fu'an tea vesicle-like nanoparticles of the present invention.

[0107] The three groups of mice were allowed to freely drink 2% DSS to establish an inflammatory bowel disease mouse model, and on the third day, each group of mice was gavaged with: physiological saline, the comparative example of squeezed Fucha vesicle-like nanoparticles, and the present invention's decocted Fucha vesicle-like nanoparticles, and each mouse was marked. The status of the mice was checked every day, and the leakage of the drinking bottle was checked.

[0108] The evaluation results of the therapeutic effects of the three groups of products on inflammatory bowel disease were obtained as follows:

[0109] During the entire experiment, the weight of each mouse was recorded to calculate the percentage of weight loss, and the feces of the mice were collected to reflect the diarrhea and bloody stools. The sum of the three scores of weight loss percentage, diarrhea score, and bloody stool score was used to calculate the disease activity index (DAI), which is the DAI value.

[0110] DAI = (body mass index + stool shape + bleeding. DAI scoring criteria: symptom scoring once a day: weight loss percentage of 0-1% is 0 points, 1%-5% is 1 point, 5%-10% is 2 points, 10%-20% is 3 points, ≥20% is 4 points; normal stool characteristics are 0 points, loose stools are 1 point, unformed stools are 2 points, and watery stools are 3 points; normal stools without blood are 0 points, stools with blood are 1 point, blood visible to the naked eye is 2 points, and heavy bleeding is 3 points.

[0111] On the ninth day, the mice were killed by cervical dislocation, and the entire colon segment was dissected out and rinsed with saline. The length of the colon was measured and photographed, and the inflammatory condition of the mice was characterized by the length of the colon.

[0112] like Figure 7As shown in the figure, the yield of the Fucha vesicle-like nanoparticles extracted by decoction provided by the present invention is 2×10 10 The number of particles / g of Fu tea, while the yield of Fu tea vesicle-like nanoparticles extracted from the control sample was 5×10 9 The number of particles per gram of Fu'er tea means that, with the same mass of Fu'er tea, the yield of the Fu'er tea vesicle-like nanoparticles extracted by decoction of the present invention is 4 times that of the Fu'er tea vesicle-like nanoparticles extracted by squeezing in a comparative example.

[0113] like Figure 8 As shown in the figure, it is shown that in the digestion process in vivo, it is faced with the degradation of strong acidic gastric juice with digestive enzymes and alkaline intestinal juice with digestive enzymes. The simulated gastric juice and simulated intestinal juice are used to simulate the digestion of the gastrointestinal tract in vitro. The figure shows that the number of particles of the vesicle-like nanoparticles extracted from Fu tea in the comparative example under normal conditions is 2.5×10 11 The particle number under simulated gastric fluid conditions was 3.7×10 11 The number of particles under simulated intestinal fluid conditions was 2.6×10 11 The results showed that the number of particles of the comparative example squeezed Fu tea vesicle-like nanoparticles increased under simulated gastric fluid, indicating that it was unstable under simulated gastric fluid.

[0114] like Fig. 9 As shown in the figure, the number of particles of the vesicle-like nanoparticles of the decoction of Fu tea in the present invention under normal conditions is 9.5×10 11 The particle number under simulated gastric fluid conditions was 9.2×10 11 The particle number under simulated intestinal fluid conditions was 8.6×10 11 The results show that the number of particles of the vesicle-like nanoparticles of the decoction of Fu tea does not change significantly under simulated gastric fluid and simulated intestinal fluid, and the stability is good.

[0115] like Fig.10 As shown, the figure shows the distribution of the comparative example squeezed Pu'er tea vesicle-like nanoparticles in the intestine, indicating that the comparative example squeezed Pu'er tea vesicle-like nanoparticles are well distributed in the colon, indicating that the particles can overcome the strong acid environment of gastric juice, the alkaline environment of intestinal fluid and the digestive effect of the gastrointestinal tract.

[0116] like Fig.11 As shown, the distribution of the vesicle-like nanoparticles of the decoction of Fu tea in the intestine is shown in the figure, showing that the vesicle-like nanoparticles of the decoction of Fu tea in the present invention are more strongly distributed in the colon. Compared with the vesicle-like nanoparticles of Fu tea squeezed in the comparative example, the vesicle-like nanoparticles of the decoction of Fu tea in the present invention are more distributed in the colon.

[0117] like Fig.12As shown, it is a schematic diagram for evaluating the effect of the comparative example squeezed Fu tea vesicle-like nanoparticles and the decocted Fu tea vesicle-like nanoparticles of the present invention in treating ulcerative colitis. The mice were allowed to freely drink 2% DSS to establish an inflammatory bowel disease mouse model, and on the third day, each group of mice was gavaged with: normal saline, the comparative example squeezed Fu tea vesicle-like nanoparticles, and the decocted Fu tea vesicle-like nanoparticles of the present invention.

[0118] like Fig.13 As shown, in order to evaluate the colon appearance of ulcerative colitis treated with the control group squeezed Fu tea vesicle-like nanoparticles and the present invention boiled Fu tea vesicle-like nanoparticles, the colon length represents the state of colon damage in ulcerative colitis mice, and it is shown that during the entire treatment period, the colon length of ulcerative colitis treated with the boiled Fu tea vesicle-like nanoparticles of the present invention was significantly longer than that of the modeling group, and the colon length of ulcerative colitis treated with the boiled Fu tea vesicle-like nanoparticles of the present invention was significantly longer than the colon length of the control group treated with the squeezed Fu tea vesicle-like nanoparticles.

[0119] like Fig.14 As shown, it is a scoring chart for evaluating the disease activity index of the comparative example of squeezing Fu tea vesicle-like nanoparticles and the present invention's decocted Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis. During the entire treatment period, the comparative example of squeezing Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis showed that the disease activity index of the comparative example of squeezing Fu tea vesicle-like nanoparticles was significantly lower than that of the modeling group, and the disease activity index of the present invention's decocted Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis was significantly lower than that of the modeling group, and the disease activity index of the present invention's decocted Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis was significantly lower than that of the comparative example of squeezing Fu tea vesicle-like nanoparticles, showing that the present invention's decocted Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis can improve the state of the entire disease process, reduce fecal bleeding and diarrhea. More importantly, the effect of the present invention's decocted Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis is better than that of the comparative example of squeezing Fu tea vesicle-like nanoparticles.

[0120] like Fig.15As shown, it is a colon length diagram for evaluating the comparative example of squeezing Fu tea vesicle-like nanoparticles and the present invention's decoction of Fu tea vesicle-like nanoparticles in the treatment of ulcerative colitis. The colon length represents the state of colon damage in mice with ulcerative colitis. The longer the length, the better the therapeutic intervention effect. During the entire treatment period, it is shown that the colon length of the ulcerative colitis treated with the decoction of Fu tea vesicle-like nanoparticles of the present invention is significantly longer than that of the modeling group, and the colon length of the ulcerative colitis treated with the decoction of Fu tea vesicle-like nanoparticles of the present invention is significantly longer than that of the comparative example of squeezing Fu tea vesicle-like nanoparticles treatment group. During the entire treatment period, the modeling group mice colon length is about 6.4 cm, the comparative example of squeezing Fu tea vesicle-like nanoparticles treatment group colon length is about 6.4 cm, and the decoction of Fu tea vesicle-like nanoparticles of the present invention is about 7.8 cm for the treatment of ulcerative colitis, showing that the decoction of Fu tea vesicle-like nanoparticles of the present invention for the treatment of ulcerative colitis can improve the state of colon damage, reduce colon damage, and show excellent therapeutic effects.

[0121] like Fig.16 As shown, the colon pathology hematoxylin-eosin staining of the decocted Fu tea vesicle-like nanoparticles of the present invention for the treatment of ulcerative colitis was performed, and the pathology of the colon tissue was detected and scored. The colon tissue of the model group showed severe destruction of the crypt structure, a decrease in goblet cells, and a large number of inflammatory cells distributed under the intestinal mucosa. Compared with the model, the appearance of the colon tissue of the control group of the extracted Fu tea vesicle-like nanoparticles and the decoction of the Fu tea vesicle-like nanoparticles of the present invention showed that the inflammation was significantly reduced. Compared with the two, the therapeutic effect of the decoction of the Fu tea vesicle-like nanoparticles of the present invention is better than that of the control group of the extracted Fu tea vesicle-like nanoparticles.

[0122] like Fig.18 The safety of the comparative example of squeezing the Pu'er tea vesicle nanoparticles and the decocted Pu'er tea vesicle nanoparticles of the present invention during oral administration was evaluated. The heart, liver, spleen, lung and kidney of mice were taken for pathological staining after oral gavage for seven days. The section results showed no obvious changes in the morphology of various tissues and organs, indicating that the treatment did not produce obvious toxic and side effects and had good biosafety.

[0123] In summary, the oral safety of the comparative example squeezed Fu tea vesicle nanoparticles and the decocted Fu tea vesicle-like nanoparticles of the present invention is higher in vivo, and the oral treatment effect of the decocted Fu tea vesicle-like nanoparticles of the present invention on ulcerative colitis is better than that of the comparative example squeezed Fu tea vesicle nanoparticles. Therefore, the decocted Fu tea vesicle-like nanoparticles of the present invention can be used as a safe strategy for the treatment of ulcerative colitis.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing pueraria tea vesicle-like nanoparticles by decoction, characterized in that: The steps include: Step 1: Preparation of Fu Tea Stock Solution: The puer tea is crushed until 60-80% of the tea powder passes through a 100-200 mesh sieve, and the sieved tea powder is brewed with water, the mass ratio of the tea powder to water is 1:6-1:10, and after boiling for at least 30 minutes, it is squeezed through a 100-200 mesh filter to obtain the puer tea stock solution; Step 2: Preparation of Fucha vesicle-like nanoparticle solution: The supernatant obtained by gradient centrifugation of the Fu Tea stock solution is placed in a container, and then centrifuged at a relative centrifugal force higher than the highest relative centrifugal force of the gradient centrifugation, and then a standard phosphate buffer with a mass ratio of 1:5 to 1:10 is added to resuspend the precipitate, so as to obtain Fu Tea vesicle-like nanoparticles; Step 3: Purification and preparation of Fucha vesicle-like nanoparticle solution: Prepare sucrose solutions with concentrations of 8%, 30%, 45%, and 60%, respectively, add the sucrose solutions into a container in order from low concentration to high concentration, then add the Fucha vesicle-like nanoparticle solution diluted 8 to 10 times with a standard phosphate buffer, wherein the volume ratio of the sucrose solution to the Fucha vesicle-like nanoparticle solution is 15:38.5 to 18:38.5, and after the formed mixed solution is centrifuged to obtain a strip containing Fucha vesicle-like nanoparticles, and suck out the strip distributed in the sucrose density layer between 8% and 30% in the container; The strip was diluted with standard phosphate buffer at a ratio of 1:6 to 1:10, and the residual sucrose solution in the strip was removed by centrifugation. The supernatant was then removed, and the precipitate in the container was diluted with standard phosphate buffer at a ratio of 1:1 to 1:5 to obtain purified Fucha vesicle-like nanoparticles.

2. The method for preparing pueraria tea vesicle-like nanoparticles by decoction as described in claim 1 is characterized in that the pueraria tea in step 1 is washed pueraria tea, and the washing process is: weigh the pueraria tea and pour it into a container, wash it with pure water at least 3 times, then wash it with distilled water at least 3 times, and then add 6 to 10 times distilled water to soak for at least 30 minutes to ensure that the leaves are completely wet.

3. The decoction preparation method of Fu'er tea vesicle-like nanoparticles as described in claim 1 is characterized in that the gradient centrifugation described in step 2 is specifically: the Fu'er tea stock solution is centrifuged at a relative centrifugal force of 500g-1500g for 10 to 20 minutes, at a relative centrifugal force of 2000g-4000g for 20 to 30 minutes, and at a relative centrifugal force of 8000g-13000g for 40 to 60 minutes to remove large particles and crude fiber in the Fu'er tea.

4. The method for preparing the pueraria tea vesicle-like nanoparticles by decoction according to claim 1, characterized in that: The relative centrifugal force higher than the highest relative centrifugal force of the gradient centrifugation in step 2 is 100000g-150000g, and the centrifugation time is 60-90min, which is used to enrich the Fucha vesicle-like nanoparticles.

5. The method for preparing the pueraria tea vesicle-like nanoparticles by decoction according to claim 1, wherein the mixed solution in step 3 is centrifuged at a relative centrifugal force of 100,000 to 150,000 g and a centrifugal time of 90 to 120 min; The relative centrifugal force of the centrifugal treatment to remove the residual sucrose in the strip is 100000-150000g, and the centrifugal time is 60-90min.

6. The method for preparing the pueraria tea vesicle-like nanoparticles by decocting as described in any one of claims 1 to 5, characterized in that the pueraria tea is pueraria tea made from dark tea or dark tea.

7. A method for preparing Fucha vesicle-like nanoparticles according to the preparation method of claim 1-6, characterized in that the particle size of the Fucha vesicle-like nanoparticles extracted by decoction is 300-480 nm, and the yield is 2×10 10 The number of particles per gram of Fu tea appears disc-shaped or tray-shaped under a transmission electron microscope and a lipid membrane structure under a scanning electron microscope.

8. An application of the Fucha vesicle-like nanoparticles as claimed in claim 7, characterized in that: Application of the Fucha vesicle-like nanoparticles in medicine for treating ulcerative colitis.

9. Oral use of the Fucha vesicle-like nanoparticles as described in claim 8 in drugs for treating ulcerative colitis.

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