Lipid vesicle-like nanoparticles of ho tea and extraction preparation method and application thereof

Abstract

The present application relates to a squeezing preparation method of the Caesalpinia minax lipid vesicle-like nanoparticles, comprising the following steps: mixing Caesalpinia minax and ultrapure water with a mass ratio of 1:6-1:10 to squeeze juice, then extruding the juice through a 100-200 mesh filter to obtain a Caesalpinia minax raw solution; then, gradient centrifuging the Caesalpinia minax raw solution to obtain a supernatant; finally, centrifuging the supernatant at a relative centrifugal force higher than the highest relative centrifugal force of the gradient centrifuging, then adding a standard phosphate buffer with a mass ratio of 1:8-1:10 to resuspend and precipitate, thereby obtaining the Caesalpinia minax lipid vesicle-like nanoparticles. The present application also provides the Caesalpinia minax lipid vesicle-like nanoparticles obtained by the method and the application of the Caesalpinia minax lipid vesicle-like nanoparticles in the treatment of ulcerative colitis. The present application overcomes the seasonal influence of the preparation of the existing tea lipid vesicle-like nanoparticles, greatly reduces the cost and controls the purity and quality, can solve the intestinal ecological imbalance problems such as mucosal epithelial repair and barrier homeostasis, has good safety and does not produce side effects, and is also a potential treatment for gastrointestinal disease treatment.

Description

Technical Field

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

[0002] Fu brick tea, a specialty product of dark tea among the six major tea categories, is a post-fermented tea, and also a fully fermented tea. For the past 600 years, Fu brick tea has been produced in Jingyang, Shaanxi Province. It is a type of dark tea fermented by *Aspergillus cristatus*, using processes such as steaming, piling, pressing, fermentation, and drying. For centuries, people have maintained the tradition of brewing it before drinking. During the production process, catechin derivatives, flavonoids and their glycosides, phenolic acids, 30 kinds of alkaloids, and terpenes are formed. Fu brick tea also has effects such as inflammatory bowel disease, lowering blood sugar and lipids, immune regulation, and anti-obesity.

[0003] Intercellular communication via the secretion of various signaling molecules is a crucial function in all multicellular organisms. In most eukaryotic cells, the release of extracellular vesicles is one of the mechanisms by which secreted components exert a key influence on neighboring or distant cells. The general term extracellular vesicles (EVs) is accepted to refer to these membranous vesicles released from the cell. Different cellular origins and characteristics define various EV subtypes, such as microvesicles, exosomes, and extracellular vesicles.

[0004] Currently, it is generally believed that all cells in plants secrete extracellular vesicles. Extracellular vesicles isolated from damaged plant tissues are named plant lipid vesicle-like nanoparticles. These are a type of membranous vesicle rich in various bioactive components, such as lipids, proteins, RNA, and potent small molecules. Plant-derived lipid vesicle-like nanoparticles offer several significant advantages as therapeutic agents. First, lipid vesicle-like nanoparticles from most edible plants have been shown to have no detectable toxicity or immunogenicity. Second, their environmental friendliness is guaranteed due to their green and sustainable plant origin. Third, plants are relatively easy to obtain. Fourth, plants do not contain human pathogens, potentially offering greater safety.

[0005] Ulcerative colitis is associated with intestinal mucosal inflammation, persistent epithelial damage, and dysbiosis. Impaired anti-inflammatory capacity is characterized by factors such as genetic susceptibility, environmental factors, and gut microbiota dysfunction.

[0006] First-line treatments for ulcerative colitis include aminosalicylate, immunosuppressants, and corticosteroids. Aminosalicylates can reduce inflammation, thus maintaining remission levels in moderate to severe IBD. Immunomodulatory drugs (anti-TNF-α agents, T-cell activation blockers, anti-adhesion agents, etc.) can cause related non-targeted systemic side effects, and long-term use may lead to serious complications. Most drug-based interventions lack specificity and have side effects. Drug therapy primarily controls disease symptoms (such as rectal bleeding, bloody diarrhea, abdominal cramps and pain) without addressing underlying gut dysbiosis issues such as mucosal epithelial repair and barrier homeostasis.

[0007] The invention application with publication number CN114642700A, entitled "Application of tea exosomes in the preparation of drugs for protecting the intestinal barrier and treating irritable bowel syndrome", describes the extraction of tea exosomes for oral administration using an extraction method. The method involves washing freshly picked tea leaves with phosphate buffer, grinding them into a homogenate, centrifuging them at differential speed, and filtering them through a 0.2-0.4 μm filter membrane to obtain tea exosomes.

[0008] The invention application CN116891850A, entitled "Tea exosome miRNA for regulating macrophage polarization, method of obtaining it and application", uses an extraction method to extract lipid vesicle-like nanoparticles from tea leaves for the regulation of macrophage polarization, and the tea exosome miRNA is obtained from fresh tea leaves.

[0009] It is evident that the extraction of lipid vesicle-like nanoparticles from tea leaves is all done by simply centrifuging fresh tea leaves after pressing. Existing technologies require a high degree of tea freshness. Since fresh tea leaves are a seasonal crop, long-term preservation is costly, and the single preparation method inevitably has a significant impact on the cost and purity of lipid vesicle-like nanoparticles.

[0010] Meanwhile, the application of Fuzhuan tea lipid vesicle-like nanoparticles in gastrointestinal treatment has only been mentioned, without further in-depth research. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying lipid vesicle-like nanoparticles obtained from processed Fu brick tea, and to apply the nanoparticles in a drug for treating ulcerative colitis.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for extracting and preparing lipid vesicle-like nanoparticles from Fuzhuan tea, comprising the following steps:

[0013] Fu brick tea and ultrapure water are mixed in a mass ratio of 1:6 to 1:10, juiced, and then squeezed through a 100-200 mesh filter to obtain the original Fu brick tea liquid.

[0014] Then, the original Fu brick tea liquid was subjected to gradient centrifugation to obtain the supernatant;

[0015] Finally, the supernatant was centrifuged at a relative centrifugal force higher than the highest gradient centrifugation force, and then resuspended in a standard phosphate buffer solution with a mass ratio of 1:8 to 1:10 to obtain Fuzhuan tea lipid vesicle-like nanoparticles.

[0016] Furthermore, the Fu brick tea is rinsed before being mixed with ultrapure water. The specific rinsing process is as follows:

[0017] Pour the Fu brick tea into a container and rinse it at least 3 times with purified water, then rinse it at least 3 times with distilled water. After that, soak it in 6 to 10 times its volume of distilled water for at least 30 minutes to ensure that the leaves are completely moistened.

[0018] Furthermore, the mixing and juicing of the Fu brick tea and ultrapure water is carried out in a juicer with a juicing power of 200-1000w and a juicing time of at least 2 minutes, so that the Fu brick tea is completely broken down.

[0019] Furthermore, the specific steps for gradient centrifugation of the Fu brick tea extract are as follows:

[0020] The original Fu brick tea liquid was poured into a centrifuge bottle and centrifuged sequentially at a relative centrifugal force of 1000-2000g for 10-20 min, 3000-6000g for 20-40 min, and 10000-12000g for 40-60 min to obtain the supernatant, which removes large particles and coarse fibers from the Fu brick tea. Further centrifugation of the supernatant at a relative centrifugal force of 130000-150000g for 60-70 min yielded Fu brick tea lipid vesicle-like nanoparticles.

[0021] Furthermore, the aforementioned Fu tea is either Fu tea or black tea made from dark tea.

[0022] The present invention also provides a lipid vesicle-like nanoparticle of Fu tea obtained by the preparation method described above. The particle size of the Fu tea lipid vesicle-like nanoparticle is 180-380 nm, the yield is 0.3-0.8 mg / g of Fu tea, and it appears as a disc or tea tray shape under transmission electron microscopy and as a lipid membrane structure under scanning electron microscopy.

[0023] The lipid components in the Fuzhuan tea lipid vesicle-like nanoparticles include: phosphatidylcholine, accounting for 15-20% of the total lipids; phosphatidylethanolamine, accounting for 16-25% of the total lipids; phosphatidylglycerol, accounting for 6-10% of the total lipids; fatty acids, accounting for 6-9% of the total lipids; and phosphatidic acid, accounting for 7-10% of the total lipids. These lipids are used to ensure the biotherapeutic effects of the lipid vesicle nanoparticles and are considered a key characteristic of the lipid vesicle nanoparticles.

[0024] The present invention also provides the application of the aforementioned Fuzhuan tea lipid vesicle-like nanoparticles in a drug for treating ulcerative colitis.

[0025] The beneficial effects of this invention are as follows: Through the technical method of this invention, lipid vesicle-like nanoparticles for processing tea are used for the first time, namely Fuzhuan tea lipid vesicle-like nanoparticles, and the extraction of Fuzhuan tea vesicle-like nanoparticles is realized. This method overcomes the seasonal influence of existing tea lipid vesicle-like nanoparticle preparations, is easy to preserve, significantly reduces costs, and allows for controllable purity and quality. In addition, the preparation method is simple and the purification is not high.

[0026] Meanwhile, this study is the first to propose the effect of Fuzhuan tea lipid vesicle-like nanoparticles on the treatment of inflammatory bowel disease, providing a new treatment strategy for the current treatment of inflammatory bowel disease, improving the symptoms of ulcerative colitis, and solving intestinal ecological imbalance problems such as mucosal epithelial repair and barrier homeostasis without producing side effects. It also provides a potential treatment method for gastrointestinal diseases. Attached Figure Description

[0027] Figure 1 The particle size distribution of the lipid vesicle-like nanoparticles from Fuzhuan tea provided by this invention is shown in the diagram.

[0028] Figure 2 Potential detection diagram of the lipid vesicle-like nanoparticles of Fuzhuan tea provided by the present invention;

[0029] Figure 3 Transmission electron microscopy image of the lipid vesicle-like nanoparticles of Fuzhuan tea provided by the present invention;

[0030] Figure 4 Scanning electron microscope image of the lipid vesicle-like nanoparticles of Fuzhuan tea provided by the present invention;

[0031] Figure 5 This is a graph showing the yield detection of lipid vesicle-like nanoparticles from Fuzhuan tea provided by the present invention.

[0032] Figure 6 The lipid detection diagram of the lipid vesicle-like nanoparticles of Fuzhuan tea provided by the present invention;

[0033] Figure 7 A schematic diagram illustrating the therapeutic effect of the Fuzhuan tea lipid vesicle-like nanoparticles provided by this invention on ulcerative colitis;

[0034] Figure 8 The image shows the weight change assessment after treatment of ulcerative colitis using the lipid vesicle-like nanoparticles of Fuzhuan tea provided by this invention.

[0035] Figure 9 A disease activity index assessment diagram for the use of Fuzhuan tea lipid vesicle-like nanoparticles provided by the present invention after treatment of ulcerative colitis;

[0036] Figure 10 The colon length diagram after treatment of ulcerative colitis using the Fuzhuan tea lipid vesicle-like nanoparticles provided by this invention.

[0037] Figure 11 The image shows the results of hematoxylin-eosin staining of the colon after treatment of ulcerative colitis using the lipid vesicle-like nanoparticles of Fuzhuan tea provided by this invention.

[0038] Figure 12 The image shows the AstraZeneca blue staining results of colon pathology after treatment of ulcerative colitis using the Fuzhuan tea lipid vesicle-like nanoparticles provided by this invention.

[0039] Figure 13 This is a diagram illustrating the safety assessment of the body during oral administration of Fuzhuan tea lipid vesicle-like nanoparticles. Detailed Implementation

[0040] 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.

[0041] To achieve the above objectives, the present invention provides the following specific embodiments:

[0042] Example 1: A method for extracting and preparing lipid vesicle-like nanoparticles from Fuzhuan tea, comprising the following steps:

[0043] Pour the Fu tea into a container. Fu tea is made from black tea. Rinse it at least 3 times with purified water, then rinse it at least 3 times with distilled water. After that, soak it in 6 to 10 times its volume of distilled water for at least 30 minutes to ensure that the leaves are completely moistened.

[0044] Next, the Fu tea and ultrapure water are mixed in a mass ratio of 1:6 to 1:10 and then juiced. The juicing is carried out in a juicer with a power of 200 to 1000w and a juicing time of at least 2 minutes to completely break down the Fu tea. The juice is then squeezed through a 100 to 200 mesh filter to obtain the original Fu tea liquid.

[0045] Then, the Fu brick tea extract was subjected to gradient centrifugation to obtain the supernatant, specifically:

[0046] Pour the original Fu brick tea liquid into a centrifuge bottle and centrifuge sequentially at a relative centrifugal force of 1000-2000g for 10-20 min, 3000-6000g for 20-40 min, 10000-12000g for 40-60 min, and 100000-150000g for 70-90 min to obtain the supernatant. This process removes large particles and coarse fibers from the Fu brick tea, resulting in high-purity Fu brick tea lipid vesicle-like nanoparticles.

[0047] Finally, the supernatant was centrifuged at a relative centrifugal force of 130,000–150,000 g for 60–70 min, and then resuspended in a standard phosphate buffer solution at a mass ratio of 1:8–1:10 to obtain the lipid vesicle-like nanoparticles of Fuzhuan tea.

[0048] Example 2: As Figures 1 to 6 As shown, the present invention also provides a Fu tea lipid vesicle-like nanoparticle obtained by the preparation method described above. The Fu tea lipid vesicle-like nanoparticle has a particle size of 180-380 nm and a yield of 0.3-0.8 mg / g of Fu tea. It appears as a disc or saucer shape under a transmission electron microscope and as a lipid membrane structure under a scanning electron microscope.

[0049] To further illustrate the effects of the present invention, the following experimental examples are provided:

[0050] Take 50g of Fu brick tea, wash it, and put it into a juicer. Add 300mL of ultrapure water to the juicer and juice for 2 minutes. Then, squeeze the juice through a filter to obtain the original liquid. Pour the original liquid into a centrifuge bottle and centrifuge according to the following gradient program: 1000g for 10 minutes, 3000g for 20 minutes, and 10000g for 40 minutes. After gradient centrifugation, pour the supernatant into an ultracentrifuge tube, balance it, and then centrifuge at 150000g for 70 minutes. After centrifugation, add 5mL of standard phosphate buffer to resuspend the precipitate, thus obtaining Fu brick tea lipid vesicle-like nanoparticles.

[0051] Then, the lipid vesicle-like nanoparticles of Fu brick tea were characterized, and the specific steps were as follows:

[0052] ① Determine its particle size and potential:

[0053] Take 20 μL of sample, dilute it to 2 mL, and measure the particle size and potential using a Bruker nanoparticle size analyzer; Figure 1 As shown in the figure, the particle size of the lipid vesicle-like nanoparticles of Fuzhuan tea prepared by the method of this invention was detected by a nanoparticle size analyzer to be 281.9 nm, and the polydispersity index was 0.219; Figure 2As shown in the figure, the potential of the lipid vesicle-like nanoparticles of Fuzhuan tea detected in the figure is -15.9mV, which shows that the lipid vesicle-like nanoparticles of Fuzhuan tea provided by the present invention do have a typical particle size distribution of nanoparticles.

[0054] ② Transmission electron microscopy characterization:

[0055] Take 30 μL of the well-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. Figure 3 As shown, the transmission electron microscopy (TEM) morphology of lipid vesicle-like nanoparticles in Fuzhuan tea was examined. Under TEM, they appeared as disc-shaped or saucer-shaped particles with a particle size of about 200 nm.

[0056] ③ Scanning electron microscopy characterization: 10 μL of the mixed sample was dropped onto a silicon wafer, allowed to air dry, and observed under a JSM-7610FPLUS scanning electron microscope. Figure 4 As shown in the figure, the scanning electron microscope (SEM) image shows the morphology of lipid vesicle-like nanoparticles in Fu brick tea. It can be seen that the lipid membrane structure is visible under the SEM, with a particle size of about 200 nm.

[0057] ④ Production detection:

[0058] The yield was determined by BCA. The product obtained above was diluted to a certain proportion, and BCA working solution was prepared according to the serial dilution of bovine serum albumin (BSA) standard (specific operation according to the kit instructions). Protein standards of various dilution concentrations and Fuzhuan tea lipid vesicle-like nanoparticles were added to microplates or test tubes, and BCA working solution (proportion according to the kit instructions) was added and mixed well. After sealing, the mixture was incubated at 37℃ for 30-60 min and cooled to room temperature. Using a blank as a control, the absorbance of the sample at 562 nm or near that wavelength was measured. The absorbance of each standard and the protein sample at 562 nm was subtracted from the average absorbance of the blank standard at 562 nm for quantification. The ratio of the measured mass of Fuzhuan tea vesicle-like nanoparticles to the mass of Fuzhuan tea was used as the yield of Fuzhuan tea lipid vesicle-like nanoparticles.

[0059] like Figure 5 As shown in the figure, the yield of lipid vesicle-like nanoparticles in Fuzhuan tea was detected, and the yield was 0.498 mg / g tissue, which is a good yield.

[0060] ⑤ Lipid identification

[0061] 100 μL of Fu brick tea lipid vesicle-like nanoparticles were added to a glass centrifuge tube with a polytetrafluoroethylene (PTFE) liner. 0.75 mL of pre-chilled methanol was added, and the mixture was vortexed. 2.5 mL of pre-chilled methyl butyl ether (MTBE) was added, and the mixture was incubated on a shaker at room temperature for 1 h. 0.625 mL of mass spectrometry-grade water was added and mixed to separate the organic phases. After incubation at room temperature for 10 min, the mixture was centrifuged at 1000 g for 10 min. The upper organic phase (MTBE) was collected, and the lower layer (water and methanol) was extracted again with 1 mL of a mixed solvent (methyl tert-butyl ether / methanol / water (10:3:2.5, v / v / v)). The upper organic phase was collected again. The two collected organic phases were concentrated by nitrogen blowing. The solutions were redissolved in 100 μL of isopropanol and then analyzed by LC-MS / MS.

[0062] like Figure 6 As shown in the figure, the lipid components of the lipid vesicle-like nanoparticles of Fuzhuan tea were detected. The lipid component analysis showed that the main lipid components were phosphatidylcholine (PC, accounting for 14.96% of the total lipids), phosphatidylethanolamine (PE, accounting for 11.16% of the total lipids), phosphatidylglycerol (PG, accounting for 7.60% of the total lipids), fatty acids (FA, accounting for 7.13% of the total lipids) and phosphatidic acid (PA, accounting for 6.18% of the total lipids).

[0063] Example 4: The present invention also provides the application of the aforementioned Fuzhuan tea lipid vesicle-like nanoparticles in a drug for treating ulcerative colitis.

[0064] To illustrate the application of the lipid vesicle-like nanoparticles obtained by this invention, the following experimental evidence is provided:

[0065] like Figure 7-12 As shown, a model of inflammatory bowel disease is established:

[0066] Seven-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 two groups: the DSS model group and the Fuzhuan tea lipid vesicle-like nanoparticle group.

[0067] Mice in the DSS modeling group were administered saline via gavage from day 0 to 10, while mice in the Fuzhuan tea lipid vesicle-like nanoparticle group were administered Fuzhuan tea lipid vesicle-like nanoparticles. From day 11 to 17, an inflammatory bowel disease mouse model was established by allowing free access to 2.5% DSS; each mouse was marked. The mice's condition was monitored daily, and leaks in the drinking bottles were checked.

[0068] 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 stools.

[0069] The Disease Activity Index (DAI) is calculated by summing the scores of three factors: percentage of body mass index (BMI), diarrhea score, and bloody stool score. The DAI value is calculated as follows: DAI = (BMI + Stool shape + Bleeding status).

[0070] DAI scoring criteria: Symptoms are scored once a day: 0-1% weight loss is 0 points, 1%-5% is 1 point, 5%-10% is 2 points, 10%-20% is 3 points, and ≥20% is 4 points; No abnormal stool characteristics are 0 points, loose stool is 1 point, unformed stool is 2 points, and watery stool is 3 points; Normal stool without bloodstains is 0 points, blood streaks in stool are 1 point, visible blood is 2 points, and massive bleeding is 3 points.

[0071] On day 18, the mice were dissected and the entire colon was removed. After rinsing with saline, the colon length was measured and photographed. The mice's weight change, disease activity index, and colon length are all important indicators of colitis.

[0072] Figure 7 This diagram illustrates the modeling of Fuzhuan tea lipid vesicle-like nanoparticles for the treatment of ulcerative colitis. Mice were acclimatized for seven days and then administered Fuzhuan tea lipid vesicle-like nanoparticles by gavage for ten days before the model was re-established to evaluate its therapeutic efficacy.

[0073] Figure 8 The image shows the weight change in patients with ulcerative colitis treated with Fuzhuan tea lipid vesicle-like nanoparticles. Throughout the treatment period, the weight loss in the Fuzhuan tea lipid vesicle-like nanoparticle treatment group was approximately 3%, while the weight loss in the model group was 12%. This indicates that Fuzhuan tea lipid vesicle-like nanoparticle treatment can inhibit weight loss and improve the symptoms of weight loss in patients with ulcerative colitis.

[0074] Figure 9 The scoring chart shows the disease activity index of ulcerative colitis treated with Fuzhuan tea lipid vesicle-like nanoparticles. Throughout the treatment phase, the disease activity index of ulcerative colitis treated with Fuzhuan tea lipid vesicle-like nanoparticles was significantly lower than that of the model group, indicating that Fuzhuan tea lipid vesicle-like nanoparticles can improve the condition of ulcerative colitis throughout the disease process and reduce fecal bleeding and diarrhea.

[0075] Figure 10This image shows the colon length in mice with ulcerative colitis treated with Fuzhuan tea lipid vesicle-like nanoparticles. Colon length represents the state of colon damage in ulcerative colitis mice; a longer length indicates a better therapeutic effect. Throughout the treatment phase, the colon length treated with Fuzhuan tea lipid vesicle-like nanoparticles was significantly longer than that in the model group. The colon length in the Fuzhuan tea lipid vesicle-like nanoparticle treatment group was approximately 6.4 cm, while the model group showed a decrease of approximately 5.5 cm. This demonstrates that Fuzhuan tea lipid vesicle-like nanoparticles can improve the state of colon damage and reduce colonic injury, exhibiting excellent therapeutic effects.

[0076] Figure 11 The colonic pathology of the colon was examined using hematoxylin-eosin staining to detect and score the pathological features of the colonic tissue in the treatment of ulcerative colitis with Fuzhuan tea lipid vesicle-like nanoparticles. In the model group, the intestinal epithelium and crypt structures were severely damaged, almost disappearing, with a large number of inflammatory cells distributed in the submucosa and significant swelling throughout the entire intestinal tissue. The Fuzhuan tea lipid vesicle-like nanoparticles protected the intestinal epithelial cells and crypts, significantly reducing inflammatory infiltration in the colonic tissue, and resulting in a marked improvement in colonic tissue damage.

[0077] Figure 12 Goblet cells and their secreted mucus form an important barrier, preventing pathogens from invading the mucosa and causing intestinal inflammation. Goblet cells play an active role in maintaining intestinal immunity and mucosal homeostasis. Abnormal proliferation and differentiation of goblet cells, as well as insufficient mucin synthesis and secretion, lead to intestinal mucosal barrier dysfunction. This study assessed mucin expression in colonic tissue using AstraZeneca blue staining. AstraZeneca blue stains acidic mucin blue. Results showed that the model group had significantly fewer goblet cells and mucin levels than the Fu brick tea lipid vesicle-like nanoparticle group, indicating that Fu brick tea lipid vesicle-like nanoparticles can significantly improve goblet cell damage in colitis.

[0078] As shown in the figures above, the Fu brick tea lipid vesicle-like nanoparticles provided by this invention have the potential to prevent weight loss. Furthermore, the DAI scores of mice administered Fu brick tea lipid vesicle-like nanoparticles via gavage were significantly lower than those of the model group. The gavage administration of Fu brick tea lipid vesicle-like nanoparticles significantly prevented colonic shortening in the mice. In summary, gavage administration of Fu brick tea lipid vesicle-like nanoparticles has a good anti-inflammatory effect and can effectively improve weight loss and rectal bleeding during the course of ulcerative colitis, providing an effective treatment for ulcerative colitis.

[0079] like Figure 13The safety of oral administration of Fuzhuan tea lipid vesicle-like nanoparticles was evaluated in mice. After seven days of oral gavage, heart, liver, spleen, lungs, and kidneys were collected for pathological staining. The sectioning results showed no significant morphological changes in any of the organs, indicating that the treatment did not produce significant toxic side effects and demonstrated good biosafety. In conclusion, oral administration of Fuzhuan tea lipid vesicle-like nanoparticles has a high in vivo safety profile and can be considered a safe strategy for the treatment of ulcerative colitis.

[0080] 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 extracting and preparing lipid vesicle-like nanoparticles from Fuzhuan tea, characterized in that, Includes the following steps: Fu brick tea and ultrapure water are mixed in a mass ratio of 1:6 to 1:10 and then juiced. The juice is then squeezed through a 100-200 mesh filter to obtain the original Fu brick tea liquid. The original Fu brick tea liquid is then subjected to gradient centrifugation to obtain the supernatant. The specific steps of gradient centrifugation are: Pour the original Fu brick tea liquid into a centrifuge bottle and centrifuge sequentially at a relative centrifugal force of 1000-2000g for 10-20 minutes, at a relative centrifugal force of 3000-6000g for 20-40 minutes, and at a relative centrifugal force of 10000-12000g for 40-60 minutes to obtain the supernatant, which removes large particles and coarse fibers from the Fu brick tea. Finally, the supernatant was centrifuged at a relative centrifugal force higher than the highest gradient centrifugation force, and then resuspended in a standard phosphate buffer solution with a mass ratio of 1:8 to 1:10 to obtain Fuzhuan tea lipid vesicle-like nanoparticles. The lipid vesicle-like nanoparticles of the Fu tea have a particle size of 180-380 nm and a yield of 0.3-0.8 mg / g of Fu tea. They appear as discs or saucers under transmission electron microscopy and as lipid membrane structures under scanning electron microscopy.

2. The method for extracting and preparing lipid vesicle-like nanoparticles from Fuzhuan tea as described in claim 1, characterized in that, The Fu brick tea was rinsed before being mixed with ultrapure water. The specific rinsing process was as follows: Pour the Fu brick tea into a container and rinse it at least 3 times with purified water, then rinse it at least 3 times with distilled water. After that, soak it in 6 to 10 times its volume of distilled water for at least 30 minutes to ensure that the leaves are completely moistened.

3. The method for extracting and preparing lipid vesicle-like nanoparticles from Fuzhuan tea as described in claim 1, characterized in that, The juicing of the Fu brick tea and ultrapure water is carried out in a juicer with a juicing power of 200-1000w and a juicing time of at least 2 minutes to completely break down the Fu brick tea.

4. The method for extracting and preparing lipid vesicle-like nanoparticles from Fuzhuan tea as described in claim 1, characterized in that, The supernatant was centrifuged at a relative centrifugal force of 130,000–150,000 g for a centrifugation time of 60–70 min.

5. An application of the lipid vesicle-like nanoparticles of Fu brick tea obtained by the method according to any one of claims 1-4, characterized in that, The application of the Fuzhuan tea lipid vesicle-like nanoparticles in the preparation of drugs for treating ulcerative colitis.

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