Fuzhuan tea vesicle-like nanoparticles, and a decocting preparation method and application thereof

Abstract

The present application relates to a kind of decocting preparation methods of Fuzhuan tea vesicle-like nanoparticles, including the preparation step of Fuzhuan tea stock solution, the preparation step of Fuzhuan tea vesicle-like nanoparticles and the purification preparation step of Fuzhuan tea vesicle-like nanoparticles.Simultaneously the present application also provides the Fuzhuan tea vesicle-like nanoparticles prepared by the method and its application.The tea raw material source used in the present application is not affected by season, completely without using organic reagent, avoid the harm caused to human body by environmental pollution and organic reagent residue, make that preparation cost is greatly reduced, the Fuzhuan tea vesicle-like nanoparticles has the characteristics of anti-gastrointestinal tract digestion, good in intestinal distribution, good safety, prove that the Fuzhuan tea vesicle-like nanoparticles of the present application can better improve the symptoms of colitis, can be applied to oral administration in the treatment of gastrointestinal diseases, simultaneously also realizes the increment of Fuzhuan tea vesicle nanoparticle yield.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to tea vesicle-like nanoparticles, their preparation methods, and applications. Background Technology

[0002] Fu brick tea is one of the treasures of the ancient Silk Road in China. It is a type of fermented dark tea. For centuries, people have been drinking Fu brick tea by boiling it in water.

[0003] Extracellular vesicles (EVs) are lipid-binding vesicles secreted into the extracellular space by all types of cells. EVs are broadly classified into three main types: exosomes (30-150 nm), microvesicles (150-1000 nm), and apoptotic bodies (1-5 μm).

[0004] Ulcerative colitis (IBD) is an idiopathic chronic inflammatory disease with a relatively high global incidence. Current treatments, such as 5-aminosalicylic acid (5-ASA), immunosuppressants (e.g., methotrexate), and biologics (e.g., infliximab, TNF-α), primarily target single-factor responses to IBD, resulting in less than ideal clinical efficacy. Furthermore, these drugs often lack specific targeting, have insufficient intestinal retention time, and are accompanied by systemic adverse reactions, leading to low remission rates in patients. Therefore, a synergistic intervention strategy is needed to restore intestinal metabolic balance.

[0005] Oral administration is the preferred route of drug delivery, but it presents significant challenges. Due to the harsh gastrointestinal environment, drugs are difficult to fully absorb through the intestinal wall. For many years, researchers have been exploring the use of functionalized nanomedicine delivery systems to improve the bioavailability of drugs after oral administration, thereby addressing the current limitations of targeted drug therapy for ulcerative colitis and reducing side effects.

[0006] Previous studies have shown that mammalian-derived exosomes can be used to treat ulcerative colitis, but their application is limited by low yield and difficulties in large-scale production. Recent studies have discovered plant-derived vesicle-like nanoparticles, which are a cutting-edge, harmless, environmentally friendly, and powerful nanocarrier, with lower immunogenicity and harmfulness than mammalian-derived exosome particles.

[0007] For example, the Chinese patent application CN117982551A, entitled "Therapeutic use of Bidens pilosa extracellular vesicles for inflammatory bowel disease", uses an extraction method to extract fresh plant vesicle-like nanoparticles for oral administration to inflammatory bowel disease. However, the Bidens pilosa used requires high freshness, and the preparation method mainly uses extraction, which will inevitably have a great impact on the cost and purity of the vesicle-like nanoparticles.

[0008] Currently established methods for preparing vesicle-like nanoparticles from tea leaves include extraction followed by ultrafiltration. Chinese patent application CN117100700A, entitled "A Fusion Nanovesicle and Its Preparation Method and Application," employs an extraction method and sucrose gradient purification for extracting vesicle-like nanoparticles from tea leaves to construct fusion vesicle-like nanoparticles. Its preparation requires the use of fresh tea leaves.

[0009] Existing methods for preparing Fu brick tea extracts often rely on large amounts of organic reagents, resulting in cumbersome processes that easily lead to pollution and organic reagent residues, posing potential harm to the human body. Chinese patent application CN107467277A, entitled "A Fu brick tea powder for regulating gastrointestinal function," describes a method for preparing a Fu brick tea powder for regulating gastrointestinal function by adding Fu brick tea to acidic water, boiling at 100°C for 30 minutes, filtering with a filter cloth, combining the two filtrates, concentrating under reduced pressure, decolorizing with activated carbon, centrifuging, and vacuum drying. This method relies on large amounts of organic reagents, is cumbersome, and fails to overcome the problems of organic reagent residues, environmental pollution, and potential harm to the human body. Furthermore, its absorption is characterized by poor targeting.

[0010] Therefore, existing technologies for preparing vesicle-like nanoparticles require high freshness of raw materials, and the main preparation method is extraction. This makes the cost and purity of vesicle-like nanoparticles highly susceptible to seasonal fluctuations, and it is difficult to control the quality of raw materials. More importantly, there is no research on how to increase the number of vesicle-like nanoparticles.

[0011] Meanwhile, existing Fu brick tea product extraction relies heavily on organic reagents, making it difficult to overcome the problems of organic reagent residues and pollution. Its application in intestinal treatment is only mentioned, such as irritable bowel syndrome. Furthermore, existing descriptions of tea vesicle-like nanoparticles only prove that tea vesicle-like nanoparticles have anti-inflammatory, antioxidant, and anti-tumor properties, without further research on ulcerative colitis. More importantly, the prepared extracts have poor targeting and significant side effects during oral application.

[0012] Therefore, it is urgent to develop a Fu tea vesicle-like nanoparticle that does not rely on organic reagents throughout the entire process, is green and environmentally friendly, has controllable quality, can overcome the digestion caused by strong acids and digestive enzymes when taken orally, and thus exerts a therapeutic effect on intestinal diseases through oral administration. Summary of the Invention

[0013] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying vesicle-like nanoparticles obtained from processed Fu brick tea without the need for organic reagents, and to apply the nanoparticles to the treatment of ulcerative colitis.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing Fu brick tea vesicle-like nanoparticles by decoction, comprising the following steps:

[0015] Step 1: Preparation of Fuzhuan Tea Extract:

[0016] Powdered Fu brick tea is pulverized so that at least 60-80% of the tea powder passes through a 100-200 mesh sieve. The sieved tea powder is brewed with water at a mass ratio of 1:6 to 1:10. After simmering for at least 30 minutes, the tea powder is then squeezed through a 100-200 mesh sieve to obtain the original Fu brick tea liquid.

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

[0018] The supernatant obtained by gradient centrifugation of Fu tea original liquid is placed in a container and then centrifuged with a relative centrifugal force higher than the highest gradient centrifugation force. Then, standard phosphate buffer with a mass ratio of 1:5 to 1:10 is added for resuspending and precipitation to obtain Fu tea vesicle-like nanoparticles.

[0019] Step 3: Purification and preparation of Fu brick tea vesicle-like nanoparticle solution:

[0020] Sucrose solutions with concentrations of 8%, 30%, 45%, and 60% were prepared. The sucrose solutions were added to the container sequentially from low to high concentration. Then, the Fu tea vesicle-like nanoparticle solution diluted 8 to 10 times with standard phosphate buffer was added. The volume ratio of the sucrose solution to the Fu tea vesicle-like nanoparticle solution was 15:38.5 to 18:38.5. After centrifugation, bands containing Fu tea vesicle-like nanoparticles were obtained. The bands distributed in the sucrose density layer between 8% and 30% were aspirated from the container.

[0021] The bands were diluted with standard phosphate buffer at a ratio of 1:6 to 1:10. The bands were centrifuged to remove residual sucrose solution. The supernatant was then discarded. The precipitate in the container was diluted with standard phosphate buffer at a ratio of 1:1 to 1:5 to obtain purified Fuzhuan tea vesicle-like nanoparticles.

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

[0023] Furthermore, the gradient centrifugation mentioned in step two specifically involves centrifuging the Fu brick tea liquid at a relative centrifugal force of 500g-1500g for 10-20 minutes, at a relative centrifugal force of 2000g-4000g for 20-30 minutes, and at a relative centrifugal force of 8000g-13000g for 40-60 minutes to remove large particles and coarse fibers from the Fu brick tea.

[0024] Furthermore, in step two, the relative centrifugal force of centrifugation at a rate higher than the gradient centrifugation is 100,000-150,000g, and the centrifugation time is 60-90min, which is used to enrich the vesicle-like nanoparticles of Fu brick tea.

[0025] Furthermore, the mixture mentioned in step three is centrifuged with a relative centrifugal force of 100,000 to 150,000 g and a centrifugation time of 90 to 120 min.

[0026] The relative centrifugal force for removing residual sucrose solution from the strips during centrifugation is 100,000–150,000 g, and the centrifugation time is 60–90 min.

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

[0028] This invention also provides Fu brick tea vesicle-like nanoparticles obtained by the preparation method described above, wherein the particle size of the Fu brick tea vesicle-like nanoparticles extracted by decoction is 300-480 nm, and the yield is 2 × 10⁻⁶. 10 The number of particles per gram of Fu brick tea, under transmission electron microscopy, appears as a disc or saucer shape, and under scanning electron microscopy, it shows a lipid membrane structure.

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

[0030] Furthermore, it also includes oral application in medications for treating ulcerative colitis, wherein the vesicle-like nanoparticles of the tea can overcome the strong acid environment of gastric juice, the alkaline environment of intestinal juice, and the digestive action of the gastrointestinal tract when taken orally, and can remain stably present in the human stomach and intestines.

[0031] The beneficial effects of this invention are:

[0032] (1) Vesicle-like nanoparticles were extracted from Fu tea by pressing and then purified and characterized.

[0033] (2) A novel method for extracting and characterizing nanoparticles from Fu brick tea sacs after decoction:

[0034] (3) The stability of the vesicle-like nanoparticles of Fu tea after decoction and the vesicle-like nanoparticles of Fu tea extracted in a comparative proportion to simulate gastrointestinal fluid was compared for the first time.

[0035] (4) This is the first report on the distribution of oral-extracted and boiled Fuzhuan tea vesicle-like nanoparticles in vivo.

[0036] Compared with existing technologies that focus on vesicle-like nanoparticles derived from fresh plants, the tea raw material source used in this invention is not affected by the season, which significantly reduces the preparation cost. The vesicle-like nanoparticles of Fu tea have the characteristics of resisting gastrointestinal digestion and good distribution in the gastrointestinal tract compared with existing reported extraction methods. This proves that decocting Fu tea vesicle-like nanoparticles can better improve the symptoms of colitis and can be applied to oral administration to treat gastrointestinal diseases. At the same time, it also achieves an increase in the yield of Fu tea vesicle-like nanoparticles.

[0037] The extraction method provided by this invention greatly improves the symptoms of ulcerative colitis and has great potential in the treatment of ulcerative colitis. It has high bioavailability when taken orally, is well tolerated by the gastrointestinal tract, and does not rely on organic reagents throughout the process. It overcomes the bottleneck of existing technologies and provides a new treatment strategy for the treatment of inflammatory bowel disease. Attached Figure Description

[0038] Figure 1 The figure shows the particle count detection results of nanoparticles extracted from Fu brick tea vesicles in a comparative manner.

[0039] Figure 2 Transmission electron microscopy image of vesicle-like nanoparticles extracted from Fu brick tea for comparative analysis.

[0040] Figure 3 Scanning electron microscopy images of nanoparticles extracted from Fu brick tea vesicles for comparative analysis.

[0041] Figure 4 This is a graph showing the particle count detection results of the vesicle-like nanoparticles in the decoction of Fu brick tea according to the present invention.

[0042] Figure 5 This is a transmission electron microscope image of the vesicle-like nanoparticles in the boiled Fu brick tea of ​​the present invention.

[0043] Figure 6 This is a scanning electron microscope image of the vesicle-like nanoparticles in the boiled Fu brick tea of ​​the present invention.

[0044] Figure 7 The yield evaluation graph is for the comparative extraction of Fu brick tea vesicle-like nanoparticles and the decoction of Fu brick tea vesicle-like nanoparticles according to the present invention. In the graph, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0045] Figure 8 A comparative chart to evaluate the digestibility of vesicle-like nanoparticles extracted from Fu brick tea in a comparative proportion.

[0046] Figure 9 This is a comparative graph evaluating the anti-digestion ability of the vesicle-like nanoparticles of Fu brick tea produced by this invention.

[0047] Figure 10 The gastrointestinal distribution diagram of the vesicle-like nanoparticles extracted from Fu brick tea for comparative analysis;

[0048] Figure 11 This is a gastrointestinal distribution diagram of the vesicle-like nanoparticles in the decoction of Fu brick tea according to the present invention.

[0049] Figure 12 This is a schematic diagram illustrating the comparative extraction of Fu brick tea vesicle-like nanoparticles and the use of the decoction of Fu brick tea vesicle-like nanoparticles provided by this invention for the treatment of ulcerative colitis.

[0050] Figure 13 Colonic images of the colon after treatment with ulcerative colitis, comparing the extracted Fu brick tea vesicle-like nanoparticles and the decoction of Fu brick tea vesicle-like nanoparticles provided by this invention.

[0051] Figure 14 This is a graph comparing the disease activity index of vesicle-like nanoparticles extracted from Fuzhuan tea and the vesicle-like nanoparticles of Fuzhuan tea provided by this invention after treatment of ulcerative colitis. In the graph, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0052] Figure 15 The graph shows the comparison of colon length after treatment of ulcerative colitis using vesicle-like nanoparticles extracted from Fuzhuan tea and the vesicle-like nanoparticles of Fuzhuan tea provided by this invention. In the graph, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significant difference.

[0053] Figure 16 Comparative images of hematoxylin-eosin staining of the colon after treatment of ulcerative colitis using extracted Fu brick tea vesicle-like nanoparticles and the decoction Fu brick tea vesicle-like nanoparticles provided by this invention.

[0054] Figure 17 This is a band distribution diagram of the Fu tea vesicle-like nanoparticles of the present invention;

[0055] Figure 18 This diagram illustrates the safety assessment of the body during the oral administration of Fuzhuan tea vesicle nanoparticles extracted by comparative proportion and the Fuzhuan tea vesicle nanoparticles prepared by this invention. Detailed Implementation

[0056] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0057] To achieve the above objectives, the present invention provides the following specific embodiments:

[0058] Example 1: As Figure 17 As shown, a method for preparing Fu brick tea vesicle-like nanoparticles by decoction includes the following steps:

[0059] Fu tea is a type of black tea, also known as black tea.

[0060] Step 1: Preparation of Fuzhuan Tea Extract:

[0061] Weigh out the Fu brick tea and pour it into a container. Rinse it at least 3 times with purified water, then rinse it at least 3 times with distilled water. After that, add 6 to 10 times the amount of distilled water and soak for at least 30 minutes to ensure that the leaves are completely moistened.

[0062] Powder the Fu tea into powder so that at least 60-80% of the tea powder passes through a 100-200 mesh sieve. Brew the sieved tea powder with water at a tea powder to water mass ratio of 1:6 to 1:10. After simmering for at least 30 minutes, squeeze the tea powder through a 100-200 mesh sieve to obtain the original Fu tea liquid.

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

[0064] The original Fu brick tea liquid was centrifuged at a relative centrifugal force of 500g-1500g for 10-20 min, 2000g-4000g for 20-30 min, and 8000g-13000g for 40-60 min to remove large particles and coarse fibers. The resulting supernatant was placed in a container and centrifuged at a relative centrifugal force of 100000-150000g for 60-90 min. The precipitate was then resuspended in a standard phosphate buffer solution at a mass ratio of 1:5 to 1:10 to obtain Fu brick tea vesicle-like nanoparticles.

[0065] Step 3: Purification and preparation of Fu brick tea vesicle-like nanoparticle solution:

[0066] Sucrose solutions with concentrations of 8%, 30%, 45%, and 60% were prepared and added sequentially to a container from low to high concentration. Then, a solution of Fu brick tea vesicle-like nanoparticles diluted 8-10 times with standard phosphate buffer was added. The volume ratio of the sucrose solution to the Fu brick tea vesicle-like nanoparticle solution was 15-18:38.5. The resulting mixture was centrifuged at a relative centrifugal force of 100,000-150,000 g for 90-120 minutes to obtain bands containing Fu brick tea vesicle-like nanoparticles. Bands distributed in the 8%-30% sucrose density layer within the container were aspirated. Figure 17 As shown;

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

[0068] Example 2: As Figures 1-7 As shown, the present invention also provides Fu brick tea vesicle-like nanoparticles obtained by the preparation method described above. The Fu brick tea vesicle-like nanoparticles extracted by decoction have a particle size of 300-480 nm and a yield of 2 × 10⁻⁶. 10 The number of particles per gram of Fu brick tea, under transmission electron microscopy, appears as a disc or saucer shape, and under scanning electron microscopy, it shows a lipid membrane structure.

[0069] To further illustrate the technical solution and effects of the present invention, the following experimental examples and comparative examples are provided, wherein the comparative examples are obtained by extracting Fu tea vesicle-like nanoparticle solutions, specifically:

[0070] ① Crude extraction of Fu brick tea vesicle-like nanoparticles:

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

[0072] Comparative extraction method: After washing, pour one portion into a juicer, add 300mL of ultrapure water to the juicer, juice for 2 minutes, and then squeeze through a 100-200 mesh filter to obtain the original extract of Fu brick tea.

[0073] The decoction method of this invention is as follows: another portion is pulverized using a grinder, the tea powder is passed through a 100-mesh sieve, the sieved tea powder is brewed with water, the mass ratio of tea powder to water is 1:6, after decoction for 30 minutes, it is then squeezed through a 200-mesh filter to obtain the decoction of Fu brick tea.

[0074] Two portions of Fu brick tea concentrate were poured into two centrifuge bottles and centrifuged using a gradient centrifugation program: 1000g for 10 min, 3000g for 20 min, and 10000g for 40 min, respectively. After gradient centrifugation, the supernatant was transferred to an ultracentrifuge tube, balanced, and then centrifuged at 150000g for 70 min. After centrifugation, 5 mL of phosphate buffer was added to resuspend the precipitate, which was then collected for subsequent sucrose purification.

[0075] ② The Fu brick tea vesicle-like nanoparticles from the comparative example and the experimental example of the present invention were purified respectively:

[0076] Sucrose solutions with concentrations of 8%, 30%, 45%, and 60% were prepared and slowly added to the centrifuge tubes sequentially from low to high concentration. Subsequently, a crude extract of Fu tea vesicle-like nanoparticles diluted with standard phosphate solution was added. After balancing the centrifuge tubes, the tubes were centrifuged at 150,000g for 90 minutes. Bands containing Fu tea vesicle-like nanoparticles were observed. The Fu tea vesicle-like nanoparticles were mainly distributed between the 8% and 30% bands. The bands were pipetted out and placed into 15mL centrifuge tubes for subsequent sugar removal.

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

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

[0079] 1) Nanoparticle tracking analysis (NTA) characterization to determine particle size and number: The specific steps are as follows:

[0080] ① Rinse the sample cell with deionized water; calibrate the instrument with polystyrene microspheres;

[0081] ② Wash the sample cell with 1XPBS bμffer;

[0082] ③ The sample was diluted with 1XPBSbμffer to measure particle size and number.

[0083] Comparative extraction methods: such as Figure 1 As shown, Figure 1 The paper shows a comparative analysis of the nanoparticle size distribution of Fu brick tea vesicles, with the vast majority being around 200 nm.

[0084] The decoction method of this invention is as follows: Figure 4 As shown, Figure 4The paper shows that the particle size distribution of the vesicle-like nanoparticles in the boiled Fu brick tea is mostly around 220 nm. Figure 5 The study showed that the vesicle-like nanoparticles of boiled Fu brick tea also appeared in a dish or saucer shape under a transmission electron microscope.

[0085] The yield is measured by the ratio of the number of particles to the mass of Fuzhuan tea, and the yields of the comparative extraction method and the inventive decoction method can be obtained respectively.

[0086] Yield of comparative extraction method: such as Figure 7 As shown in the comparative example, the yield of the comparative extraction of Fu brick tea vesicle-like nanoparticles is 5 × 10⁻⁶. 9 Number of particles per gram of Fu brick tea.

[0087] Invention Example: Decoction Method Yield: As shown Figure 7 The yield of the Fuzhuan tea vesicle-like nanoparticles extracted by decoction provided by this invention is 2 × 10⁻⁶. 10 The yield of the Fu tea vesicle-like nanoparticles extracted by the present invention per gram of Fu tea can be compared with that of the comparative example and the invention example to demonstrate that, using the same quality of Fu tea, the yield of Fu tea vesicle-like nanoparticles extracted by the present invention is 4 times that of the Fu 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 onto a copper grid with a carbon support film. Let it stand for 5 minutes. Use filter paper to absorb any remaining liquid from the edge of the copper grid. Add 2% phosphotungstic acid to the copper grid and stain for 30 seconds at room temperature. Use filter paper to absorb any excess stain. Allow it to air dry. Observe under a JEM-1400FLASH transmission electron microscope.

[0090] Comparative extraction methods: such as Figure 2 As shown, Figure 2 The nanoparticles of Fu brick tea extracted in a medium-to-comparative ratio appear as disc-shaped or saucer-shaped under a transmission electron microscope.

[0091] The decoction method of this invention is as follows: Figure 5 As shown in the figure, the nanoparticles resembling vesicles of the Fu brick tea produced by this invention also appear as discs or saucers under a transmission electron microscope.

[0092] 3) Scanning electron microscopy characterization:

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

[0094] Comparative extraction methods: such as Figure 3 As shown, Figure 3 The paper shows the lipid membrane structure of vesicle-like nanoparticles extracted from Fu brick tea under a comparative ratio under a scanning electron microscope.

[0095] The decoction method of this invention is as follows: Figure 6 As shown, Figure 6 The paper demonstrates that the vesicle-like nanoparticles of the decoction Fu brick tea of ​​the present invention exhibit a lipid membrane structure under a scanning electron microscope.

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

[0097] To further illustrate the technical application effects of the present invention, the following experimental evidence is provided, in which the results of comparative experiments using the extraction method of Fu brick tea are compared with the effects of the present invention:

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

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

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

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

[0102] 3) Prepare a solution of the fluorescent dye DiR. DiR is a lipophilic fluorescent dye frequently used to stain cell membranes and other lipid-soluble biological structures. Because the near-infrared fluorescence of DiR can penetrate cells and tissues, it is also frequently used in small animal in vivo imaging techniques. This was used to visualize the in vivo distribution of Fuzhuan tea vesicle-like nanoparticles. Weigh 2 mg of DiR, add 197 μL of dimethyl disulfone 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 obtain the working solution.

[0103] 4) The above-prepared DiR is combined with the comparative extracted Fu tea vesicle-like nanoparticles and the decoction Fu tea vesicle-like nanoparticles of the present invention, so that the concentration of the comparative extracted 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 dehaired with hair removal cream, and then 0.2 mL of DiR-bound comparative extracted Fu tea vesicle-like nanoparticles and 0.2 mL of DiR-bound decoction of Fu tea vesicle-like nanoparticles of this invention were administered by gavage. Six hours later, the mice were dissected and the fluorescence intensity in the gastrointestinal tract was observed using an in vivo imaging instrument.

[0105] 6) such as Figure 7-16 As shown, a model of inflammatory bowel disease is established:

[0106] Twenty 7-week-old male C57BL / 6J mice were housed at the Experimental Animal Center of the Institute of Medical Sciences, Northwestern Polytechnical University. After one week of free-range feeding, five mice were randomly selected per cage as the normal control group, and the remaining mice were randomly divided into three groups: the DSS modeling group, the comparative extraction group of Fu brick tea vesicle-like nanoparticles, and the Fu brick tea vesicle-like nanoparticle group prepared according to this invention.

[0107] Three groups of mice were used to establish an inflammatory bowel disease mouse model by freely drinking 2% DSS. On the third day, each group of mice was administered physiological saline, comparatively extracted Fu brick tea vesicle-like nanoparticles, or the decoction of Fu brick tea vesicle-like nanoparticles according to this invention by gavage. Each mouse was marked. The mice's condition was checked daily, and the water bottles were inspected for leaks.

[0108] The evaluation results of the therapeutic effects of the three groups of products on ulcerative colitis are as follows:

[0109] Throughout the experiment, the weight of each mouse was recorded to calculate the percentage of weight loss, and mouse feces were collected to reflect diarrhea and bloody stool. The Disease Activity Index (DAI) was calculated by summing the scores of the percentage of weight loss, diarrhea score, and bloody stool score; this is the DAI value.

[0110] DAI = (Body Mass Index + Stool Shape + Bleeding). DAI scoring criteria: Symptoms are scored once a day: 0-1% weight loss = 0 points, 1%-5% = 1 point, 5%-10% = 2 points, 10%-20% = 3 points, ≥20% = 4 points; No abnormal stool characteristics = 0 points, loose stool = 1 point, unformed stool = 2 points, watery stool = 3 points; Normal stool without blood = 0 points, blood streaks in stool = 1 point, visible blood = 2 points, massive bleeding = 3 points.

[0111] On the ninth day, the mice were euthanized by cervical dislocation, and the entire colon was dissected, rinsed with saline, and the colon length was measured and photographed. Colon length was used to characterize the inflammatory status of the mice.

[0112] like Figure 7As shown in the figure, the yield of the Fuzhuan tea vesicle-like nanoparticles extracted by decoction according to the present invention is 2 × 10⁻⁶. 10 The number of particles per gram of Fu brick tea, while the yield of vesicle-like nanoparticles extracted by comparative pressing of Fu brick tea is 5 × 10⁻⁶. 9 The number of particles per gram of Fu tea indicates that, using the same quality of Fu tea, the yield of Fu tea vesicle-like nanoparticles extracted by this invention is 4 times that of Fu tea vesicle-like nanoparticles extracted by proportional pressing.

[0113] like Figure 8 As shown in the figure, during the in vivo digestion process, the digestive system faces degradation from both highly acidic gastric juice (containing digestive enzymes) and alkaline intestinal juice (containing digestive enzymes). Simulated gastric and intestinal juices are used to simulate the digestive process in the in vitro gastrointestinal tract. The figure shows that the number of nanoparticles resembling vesicles extracted from Fu brick tea in a comparative proportion is 2.5 × 10⁻⁶ under normal conditions. 11 The number of particles was 3.7 × 10⁶ under simulated gastric fluid conditions. 11 The number of particles was 2.6 × 10⁶ under simulated intestinal fluid conditions. 11 The results showed that the number of nanoparticles extracted from Fu brick tea vesicles increased under simulated gastric juice conditions, indicating that they were unstable under simulated gastric juice conditions.

[0114] like Figure 9 As shown in the figure, the number of nanoparticles resembling vesicles in the decoction of Fu brick tea according to the present invention is 9.5 × 10⁻⁶ under normal conditions. 11 The number of particles was 9.2 × 10⁶ under simulated gastric fluid conditions. 11 The number of particles was 8.6 × 10⁶ under simulated intestinal fluid conditions. 11 The results showed that the number of particles in the vesicle-like nanoparticles of the Fu brick tea decoction of the present invention did not change significantly under simulated gastric juice and simulated intestinal juice, indicating good stability.

[0115] like Figure 10 As shown in the figure, the distribution of comparative extracted Fu brick tea vesicle-like nanoparticles in the intestine is displayed. The comparative extracted Fu brick 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 juice, and the digestive action of the gastrointestinal tract.

[0116] like Figure 11 As shown in the figure, the distribution of the vesicle-like nanoparticles of the Fu brick tea decocted according to the present invention in the intestine is illustrated, indicating a stronger distribution of these nanoparticles in the colon. Compared to the vesicle-like nanoparticles extracted from Fu brick tea in the comparative example, the vesicle-like nanoparticles of the Fu brick tea decocted according to the present invention are more abundant in the colon.

[0117] like Figure 12As shown in the diagram, to evaluate the efficacy of comparatively extracted Fu brick tea vesicle-like nanoparticles and the decoction of Fu brick tea vesicle-like nanoparticles of the present invention in treating ulcerative colitis, mice were used to establish an inflammatory bowel disease mouse model by freely drinking 2% DSS. On the third day, each group of mice was administered physiological saline, comparatively extracted Fu brick tea vesicle-like nanoparticles, and the decoction of Fu brick tea vesicle-like nanoparticles of the present invention by gavage.

[0118] like Figure 13 As shown, to evaluate the colonic appearance of mice with ulcerative colitis treated with comparatively extracted Fu brick tea vesicle-like nanoparticles and the present invention's decoction of Fu brick tea vesicle-like nanoparticles, colon length represents the state of colonic damage in mice with ulcerative colitis. The results show that throughout the treatment phase, the colonic length of the mice treated with the present invention's decoction of Fu brick tea vesicle-like nanoparticles was significantly longer than that of the model group, and the colonic length of the mice treated with the present invention's decoction of Fu brick tea vesicle-like nanoparticles was significantly longer than that of the comparatively extracted Fu brick tea vesicle-like nanoparticles treatment group.

[0119] like Figure 14 As shown in the figure, a scoring chart was used to evaluate the disease activity index of ulcerative colitis treated with comparatively extracted Fu brick tea vesicle-like nanoparticles and the ulcerative Fu brick tea vesicle-like nanoparticles of the present invention. Throughout the treatment phase, the disease activity index of ulcerative colitis treated with comparatively extracted Fu brick tea vesicle-like nanoparticles was significantly lower than that of the model group. The disease activity index of ulcerative colitis treated with the ulcerative Fu brick tea vesicle-like nanoparticles of the present invention was also significantly lower than that of the model group. Furthermore, the disease activity index of ulcerative colitis treated with the ulcerative Fu brick tea vesicle-like nanoparticles of the present invention was significantly lower than that of the comparatively extracted Fu brick tea vesicle-like nanoparticle group. This indicates that the ulcerative Fu brick tea vesicle-like nanoparticles of the present invention can improve the condition of ulcerative colitis throughout the disease process, reducing fecal bleeding and diarrhea. More importantly, the therapeutic effect of the ulcerative Fu brick tea vesicle-like nanoparticles of the present invention on ulcerative colitis is superior to that of the comparatively extracted Fu brick tea vesicle-like nanoparticles.

[0120] like Figure 15As shown, this diagram illustrates the colon length in mice with ulcerative colitis treated with comparatively extracted Fu brick tea vesicle-like nanoparticles and the present invention's decoction of Fu brick tea vesicle-like nanoparticles. Colon length represents the state of colon damage in mice with ulcerative colitis; a longer length indicates a better therapeutic intervention. Throughout the treatment phase, the colon length in the mice treated with the present invention's decoction of Fu brick tea vesicle-like nanoparticles was significantly longer than that in the model group. Furthermore, the colon length in the mice treated with the present invention's decoction of Fu brick tea vesicle-like nanoparticles was significantly longer than that in the comparatively extracted Fu brick tea vesicle-like nanoparticles group. Throughout the treatment phase, the colon length in the model group was approximately 6.4 cm, while the colon length in the comparatively extracted Fu brick tea vesicle-like nanoparticles group was approximately 6.4 cm, and the colon length in the mice treated with the present invention's decoction of Fu brick tea vesicle-like nanoparticles was approximately 7.8 cm. This demonstrates that the present invention's decoction of Fu brick tea vesicle-like nanoparticles can improve the state of colon damage and reduce colonic injury, exhibiting excellent therapeutic effects.

[0121] like Figure 16 As shown, the colonic pathology of the vesicle-like nanoparticles from Fu brick tea used in this invention for the treatment of ulcerative colitis was examined and scored using hematoxylin-eosin staining. The model group showed severe crypt structure destruction, reduced goblet cells, and a large number of inflammatory cells distributed in the submucosa. Compared to the model, the colonic tissue of both the comparative extraction of Fu brick tea vesicle-like nanoparticles and the group using the vesicle-like nanoparticles from the decocted Fu brick tea of ​​this invention showed significantly reduced inflammation. Therefore, the therapeutic effect of the vesicle-like nanoparticles from the decocted Fu brick tea of ​​this invention is superior to that of the comparative extraction group.

[0122] like Figure 18 The safety of the comparative extracted Fu brick tea vesicle nanoparticles and the decoction-like Fu brick tea vesicle nanoparticles of the present invention during oral administration was evaluated. After oral administration for seven days, the heart, liver, spleen, lungs and kidneys of mice were collected for pathological staining. The section results showed no significant morphological changes in the organs, indicating that the treatment did not produce obvious toxic side effects and had good biosafety.

[0123] In summary, both the comparatively extracted Fu brick tea vesicle nanoparticles and the decocted Fu brick tea vesicle-like nanoparticles of the present invention have high in vivo safety profiles when administered orally. The oral administration of the decocted Fu brick tea vesicle-like nanoparticles of the present invention is more effective than that of the comparatively extracted Fu brick tea vesicle nanoparticles in treating ulcerative colitis. Therefore, the decocted Fu brick 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing Fu brick tea vesicle-like nanoparticles by decoction, characterized in that, Includes the following steps: Step 1: Preparation of Fuzhuan Tea Extract: Powdered Fu tea is pulverized to 60-80% purity and passed through a 100-200 mesh sieve. The sieved tea powder is brewed with water at a ratio of 1:6 to 1:

10. After simmering for at least 30 minutes, the tea powder is then squeezed through a 100-200 mesh sieve to obtain the original Fu tea liquid. Step 2: Preparation of Fuzhuan tea vesicle-like nanoparticle solution: The supernatant obtained by gradient centrifugation of Fu tea original liquid is placed in a container and then centrifuged with a relative centrifugal force higher than the highest gradient centrifugation force. Then, standard phosphate buffer with a mass ratio of 1:5 to 1:10 is added for resuspending and precipitation to obtain Fu tea vesicle-like nanoparticles. Step 3: Purification and preparation of Fu brick tea vesicle-like nanoparticle solution: Sucrose solutions with concentrations of 8%, 30%, 45%, and 60% were prepared. The sucrose solutions were added to the container sequentially from low to high concentration. Then, the Fu tea vesicle-like nanoparticle solution diluted 8 to 10 times with standard phosphate buffer was added. The volume ratio of the sucrose solution to the Fu tea vesicle-like nanoparticle solution was 15:38.5 to 18:38.

5. After centrifugation, bands containing Fu tea vesicle-like nanoparticles were obtained. The bands distributed in the sucrose density layer between 8% and 30% were aspirated from the container. The bands were diluted with standard phosphate buffer at a ratio of 1:6 to 1:

10. The bands were centrifuged to remove residual sucrose solution. The supernatant was then discarded. The precipitate in the container was diluted with standard phosphate buffer at a ratio of 1:1 to 1:5 to obtain purified Fuzhuan tea vesicle-like nanoparticles.

2. The method for preparing Fu tea vesicle-like nanoparticles by decoction as described in claim 1, characterized in that the Fu tea in step one is Fu tea that has been washed, and the washing process is as follows: 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 the amount of distilled water to soak for at least 30 minutes to ensure that the leaves are completely moistened.

3. The method for preparing Fu tea vesicle-like nanoparticles by decoction as described in claim 1, characterized in that the gradient centrifugation in step two specifically involves: centrifuging the Fu tea stock solution at a relative centrifugal force of 500g-1500g for 10-20 min, at a relative centrifugal force of 2000g-4000g for 20-30 min, and at a relative centrifugal force of 8000g-13000g for 40-60 min, to remove large particles and coarse fibers from the Fu tea.

4. The method for preparing Fuzhuan tea vesicle-like nanoparticles by decoction as described in claim 1, characterized in that, In step two, the centrifugal force of 150,000 g, which is higher than the highest gradient centrifugation, is used to enrich the vesicle-like nanoparticles of Fu tea.

5. The decoction preparation method of Fuzhuan tea vesicle-like nanoparticles as described in claim 1, Its characteristic is that the mixture in step three is centrifuged with a relative centrifugal force of 100,000 to 150,000 g and a centrifugation time of 90 to 120 min; The centrifugal treatment to remove residual sucrose in the strips involves a relative centrifugal force of 100,000–150,000 g and a centrifugation time of 70–90 min.

6. The use of the Fuzhuan tea vesicle-like nanoparticles obtained by the decoction preparation method according to any one of claims 1-5 in the preparation of drugs for treating ulcerative colitis.

7. The use of the Fuzhuan tea vesicle-like nanoparticles obtained by the decoction preparation method according to any one of claims 1-5 in the preparation of oral medications for treating ulcerative colitis.

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