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

Abstract

The invention relates to a squeezing preparation method of Fuzhuan tea lipid vesicle-like nanoparticles, which comprises the following steps: mixing Fuzhuan tea and ultrapure water in a mass ratio of (1: 6)-(1: 10), squeezing juice, passing through a 100-200-mesh filter screen, and squeezing to obtain a Fuzhuan tea stock solution; then, carrying out gradient centrifugation on the Fuzhuan tea stock solution to obtain supernate; and finally, carrying out centrifugal treatment on the supernatant at a relative centrifugal force higher than the highest relative centrifugal force of gradient centrifugation, and adding a standard phosphate buffer solution with the mass ratio of 1: 8-1: 10 to carry out resuspension precipitation, so as to obtain the Fuzhuan tea lipid vesicle-like nanoparticles. The invention also provides the Fuzhuan tea lipid vesicle-like nanoparticles obtained by using the method and application of the Fuzhuan tea lipid vesicle-like nanoparticles in treatment of ulcerative colitis. According to the invention, the seasonal influence of the preparation of the existing tea lipid vesicle-like nanoparticles is overcome, the cost is greatly reduced, the purity and quality are controllable, the problems of intestinal ecological imbalance such as mucosal epithelium repair, barrier homeostasis and the like can be solved, the safety is good, no side effect is generated, and a potential treatment mode is provided for the treatment of gastrointestinal diseases.

Description

Technical Field

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

[0002] Fucha is a special product of black tea among the six major types of tea. It is a post-fermented tea and a fully fermented tea. Fucha has been produced in Jingyang, Shaanxi for the past 600 years. Fucha is a black tea fermented by Eurotium coronatum. It is made by steaming, piling, pressing, fermenting, and drying. For thousands of years, people have maintained the tradition of drinking tea after boiling. In the production process of Fucha, catechin derivatives, flavonoids and their glycosides, phenolic acids, 30 alkaloids and terpenes are formed. At the same time, Fucha also includes the effects of inflammatory bowel disease, hypoglycemic and lipid-lowering, immune regulation, and anti-obesity.

[0003] Communication between cells through the secretion of various signaling molecules is a key function of all multicellular organisms. In most eukaryotic cells, the release of extracellular vesicles is one of the mechanisms by which secretory components exert key effects on neighboring or distant cells. The general term extracellular vesicles (EVs) has been accepted to refer to these membrane-bound vesicles released from cells. Different cellular origins and characteristics define a variety of EV subtypes, such as microvesicles, exosomes, and extracellular vesicles.

[0004] At present, it is generally believed that all cells, including plant cells, secrete extracellular vesicles. Extracellular vesicles isolated from damaged plant tissues are named plant lipid vesicle-like nanoparticles, which are a class of membrane vesicles rich in various bioactive components such as lipids, proteins, RNA and effective small molecules. Plant-derived lipid vesicle-like nanoparticles have several obvious advantages as therapeutic drugs. First, lipid vesicle-like nanoparticles from most edible plants have been shown to have no detectable toxicity or immunogenicity. Second, they are environmentally friendly due to their green and sustainable plant sources. Third, plants can be obtained relatively easily. Fourth, plants do not have human pathogenic pathogens and may be safer.

[0005] Ulcerative colitis is associated with intestinal mucosal inflammation, persistent epithelial damage, and dysbiosis, characterized by impaired anti-inflammatory capacity in the context of genetic susceptibility, environmental factors, and a dysbiosis of the intestinal microbiota.

[0006] First-line treatments for ulcerative colitis include aminosalicylates, immunosuppressants, and corticosteroids. Aminosalicylates can reduce inflammation, thereby maintaining the remission level of moderate to severe IBD; immunomodulatory drugs (anti-TNF-α agents, T cell activation blockers, anti-adhesion agents, etc.) can cause related non-target systemic side effects, and long-term use may cause serious complications. Most drug-based interventions lack specificity and have side effects. Drug treatment mainly controls the symptoms of the disease (such as rectal bleeding, bloody diarrhea, abdominal cramps and pain), but does not solve the intestinal ecological imbalance problems such as mucosal epithelial repair and barrier homeostasis.

[0007] The invention application with publication number CN114642700A, "Application of tea exosomes in the preparation of drugs for protecting intestinal barrier and treating irritable bowel syndrome", adopts a squeezing method to extract oral drugs for irritable bowel syndrome. Freshly picked tea leaves are cleaned with phosphate buffer, ground into a homogenate, subjected to differential centrifugation, and filtered with a 0.2-0.4 μm filter membrane to obtain tea exosomes.

[0008] The invention application with publication number CN116891850A "Tea exosome miRNA for regulating macrophage polarization and its acquisition method and application" uses a squeezing method to extract tea lipid vesicle-like nanoparticles for regulating macrophage polarization, and the tea exosome miRNA is obtained from fresh tea leaves.

[0009] It can be seen that the extracted tea lipid vesicle-like nanoparticles are all prepared by squeezing fresh tea leaves and then taking simple centrifugation. The existing technical solutions have high requirements on the freshness of the tea leaves used. Since fresh tea leaves are a seasonal crop, the cost of long-term storage is high, and the preparation method is single, which will inevitably greatly affect the cost and purity of the lipid vesicle-like nanoparticles.

[0010] At the same time, the application of Pu'er tea lipid vesicle-like nanoparticles in gastrointestinal treatment has only been mentioned, and no further research has been done. Summary of the invention

[0011] The purpose of the present invention is to avoid the shortcomings of the prior art and provide a kind of lipid vesicle-like nanoparticles obtained from processed Fu'er tea, and apply the nanoparticles to Fu'er tea lipid vesicle-like nanoparticles in drugs for treating ulcerative colitis, as well as a preparation method and application thereof.

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

[0013] Mixing Fu tea and ultrapure water in a mass ratio of 1:6 to 1:10 to squeeze out the juice, and then passing through a 100-200 mesh filter to obtain Fu tea stock solution;

[0014] Then, the Fu tea stock solution was subjected to gradient centrifugation to obtain a supernatant;

[0015] Finally, the supernatant is centrifuged at a relative centrifugal force higher than the highest relative centrifugal force of the gradient centrifugation, and then a standard phosphate buffer having a mass ratio of 1:8 to 1:10 is added to resuspend the precipitate to obtain Fu'er tea lipid vesicle-like nanoparticles.

[0016] Furthermore, the Fu tea is washed before being mixed with ultrapure water, and the specific washing process is:

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

[0018] Furthermore, the mixed juice extraction of the Fu tea and ultrapure water is carried out in a juicer with a juice extraction power of 200-1000w and a juice extraction time of at least 2min, so that the Fu tea is completely broken.

[0019] Furthermore, the specific steps of subjecting the Fu tea stock solution to gradient centrifugation are:

[0020] The tuckahoe tea stock solution is poured into a centrifuge bottle, and centrifuged at a relative centrifugal force of 1000-2000g for 10-20min, at a relative centrifugal force of 3000-6000g for 20-40min, and at a relative centrifugal force of 10000-12000g for 40-60min to obtain the supernatant, so as to remove large particles and crude fibers in the tuckahoe tea. The supernatant is further centrifuged at a relative centrifugal force of 130000-150000g for 60-70min to obtain tuckahoe tea lipid vesicle-like nanoparticles.

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

[0022] The present invention also provides a kind of Fu'er tea lipid vesicle-like nanoparticles obtained by the preparation method. The Fu'er tea lipid vesicle-like nanoparticles have a particle size of 180-380 nm and a yield of 0.3-0.8 mg / g of Fu'er tea. They are disc-shaped or tray-shaped under a transmission electron microscope and have a lipid membrane structure under a scanning electron microscope.

[0023] The lipid components in the Fucha lipid vesicle 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, which ensures that the lipids of the lipid vesicle nanoparticles have biological therapeutic effects and serve as the key features of the lipid vesicle nanoparticles.

[0024] The present invention also provides application of the Fucha lipid vesicle-like nanoparticles in medicine for treating ulcerative colitis.

[0025] The beneficial effects of the present invention are: through the technical method of the present invention, the lipid vesicle-like nanoparticles of processed tea, namely Pu'er tea lipid vesicle-like nanoparticles, are used for the first time, and the extraction of Pu'er tea vesicle-like nanoparticles is realized, the processed tea overcomes the seasonal influence of the existing preparation of tea lipid vesicle-like nanoparticles, is easy to preserve, greatly reduces the cost, and the purity and quality are controllable, and the preparation method is single and the purity is not high.

[0026] At the same time, it is the first time that the effect of Pu'er tea lipid vesicle-like nanoparticles on the treatment of inflammatory bowel disease has been proposed, which provides a new treatment strategy for the current treatment of inflammatory bowel disease, improves the symptoms of ulcerative colitis, and can solve intestinal ecological disorders such as mucosal epithelial repair and barrier homeostasis without causing side effects, and also provides a potential treatment method for gastrointestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a particle size detection diagram of the Fu'an tea lipid vesicle-like nanoparticles provided by the present invention;

[0028] Figure 2 This is a potential detection diagram of the Fu'er tea lipid vesicle-like nanoparticles provided by the present invention;

[0029] Figure 3 A transmission electron micrograph of the Pu'er tea lipid vesicle-like nanoparticles provided by the present invention;

[0030] Figure 4 This is a scanning electron microscope image of the Fu'er tea lipid vesicle-like nanoparticles provided by the present invention;

[0031] Figure 5 This is a graph showing the yield of the Fu'an tea lipid vesicle-like nanoparticles provided by the present invention;

[0032] Figure 6 This is a lipid detection diagram of the Fu'an tea lipid vesicle-like nanoparticles provided by the present invention;

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

[0034] Figure 8 This is a graph evaluating weight changes after the Fucha lipid vesicle-like nanoparticles provided by the present invention are used to treat ulcerative colitis;

[0035] Fig. 9 A disease activity index evaluation graph after the Fucha lipid vesicle-like nanoparticles provided by the present invention are used to treat ulcerative colitis;

[0036] Fig.10 This is a graph of colon length after the Fu'er tea lipid vesicle-like nanoparticles provided by the present invention are used to treat ulcerative colitis.

[0037] Fig.11 This is a graph showing the hematoxylin-eosin staining results of colon pathology after the Fu'er tea lipid vesicle-like nanoparticles provided by the present invention are used to treat ulcerative colitis.

[0038] Fig.12 This is a graph showing the results of Astley blue staining of colon pathology after the Fu'er tea lipid vesicle-like nanoparticles provided by the present invention are used to treat ulcerative colitis;

[0039] Fig.13 This is a diagram for the safety assessment of Pu'er tea lipid vesicle-like nanoparticles during oral administration. DETAILED DESCRIPTION

[0040] 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.

[0041] In order to achieve the above object, the present invention provides the following specific implementation methods:

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

[0043] Pour the Fucha into a container. Fucha is Fucha or black tea made from dark tea. Wash it at least 3 times with purified water, then at least 3 times with distilled water, and then soak it in 6-10 times distilled water for at least 30 minutes to ensure that the leaves are completely wet.

[0044] Next, the Fu tea and ultrapure water are mixed in a mass ratio of 1:6 to 1:10 and juiced in a juicer with a juicer power of 200 to 1000 W and a juicer time of at least 2 minutes, so that the Fu tea is completely broken, and the Fu tea stock solution is obtained by squeezing through a 100 to 200 mesh filter;

[0045] Then, the Fu tea stock solution was subjected to gradient centrifugation to obtain a supernatant, specifically:

[0046] The Pu'er tea stock solution is poured into a centrifuge bottle, and centrifuged at a relative centrifugal force of 1000-2000g for 10-20min, at a relative centrifugal force of 3000-6000g for 20-40min, at a relative centrifugal force of 10000-12000g for 40-60min, and at a relative centrifugal force of 100000-150000g for 70-90min to obtain the supernatant, so as to remove large particles and crude fibers in the Pu'er tea and obtain high-purity Pu'er tea lipid vesicle-like nanoparticles.

[0047] Finally, the supernatant was centrifuged at a relative centrifugal force of 130,000 to 150,000 g for 60 to 70 min, and then a standard phosphate buffer with a mass ratio of 1:8 to 1:10 was added to resuspend the precipitate to obtain Pu'er tea lipid vesicle-like nanoparticles.

[0048] Example 2: Figures 1 to 6 As shown, the present invention also provides a kind of Pu'er tea lipid vesicle-like nanoparticles obtained by the preparation method, the Pu'er tea lipid vesicle-like nanoparticles have a particle size of 180-380nm, a yield of 0.3-0.8 mg / g Pu'er tea, and are disc-shaped or tray-shaped under a transmission electron microscope and have a lipid membrane structure under a scanning electron microscope.

[0049] In order to further illustrate the effect of the present invention, the following test examples are provided:

[0050] Take 50g of Fucha and wash it and pour it into a juicer. Add 300mL of ultrapure water to the juicer. After squeezing for 2 minutes, squeeze the stock solution with a filter. Pour the stock solution into a centrifuge bottle and centrifuge at 1000g for 10 minutes, 3000g for 20 minutes, and 10000g for 40 minutes. After the gradient centrifugation, pour the supernatant into the ultracentrifuge tube to balance, and then centrifuge at 150000g for 70 minutes. After the centrifugation is completed, add 5mL of standard phosphate buffer to resuspend the precipitate, and obtain Fucha lipid vesicle-like nanoparticles.

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

[0052] ① Determine its particle size and potential:

[0053] Take 20 μL of sample and dilute it to 2 mL, and use Bruker nanoparticle size analyzer to measure the particle size and potential; Figure 1 As shown in the figure, the particle size of the Fucha lipid vesicle-like nanoparticles prepared by the method of the present invention is 281.9 nm and the polydispersity index is 0.219. Figure 2As shown in the figure, the potential of the Pu'er tea lipid vesicle-like nanoparticles detected in the figure is -15.9mv, which shows that the Pu'er tea lipid vesicle-like nanoparticles provided by the present invention are indeed typical nanoparticles in particle size distribution.

[0054] ② Transmission electron microscopy characterization:

[0055] 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 remaining 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. Figure 3 As shown, the transmission electron microscopy morphology of Pu'er tea lipid vesicle-like nanoparticles was detected, which appeared disc-shaped or tray-shaped under a transmission electron microscope, with a particle size of about 200 nm.

[0056] ③ Scanning electron microscope characterization: Take 10 μL of the mixed sample and drop it on the silicon wafer, let it air dry naturally, and observe it under a JSM-7610FPLUS scanning electron microscope. Figure 4 As shown in the figure, the scanning electron microscopy morphology of Pu'er tea lipid vesicle-like nanoparticles is detected. It can be seen that under the scanning electron microscope, a lipid membrane structure is present and the particle size is about 200nm.

[0057] ④Production detection:

[0058] The yield is determined by BCA, the above-obtained product is diluted in a certain proportion, and the BCA working solution is prepared according to the gradient dilution of the bovine serum albumin (BSA) standard (the specific operation is carried out according to the instructions in the kit) (the specific operation is carried out according to the instructions). The protein standard and Fucha lipid vesicle-like nanoparticles of each dilution concentration are added to the microplate or test tube, and the BCA working solution (the proportion is referred to the instructions of the kit) is added respectively and mixed. After sealing, keep warm at 37°C for 30-60min and cool to room temperature. With the blank as the control, measure the absorbance of the sample at 562nm or near this wavelength. The absorbance of each standard and the protein sample to be tested at 562nm is subtracted from the average absorbance of the blank standard at 562nm for quantification. The mass ratio of the measured Fucha vesicle-like nanoparticles to the mass of Fucha is used as the yield of Fucha lipid vesicle-like nanoparticles.

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

[0060] ⑤ Lipid identification

[0061] 100 μL of Fucha lipid vesicle-like nanoparticles were added to a glass centrifuge tube with a polytetrafluoroethylene-lined cap, and 0.75 mL of pre-cooled methanol was added and vortexed. 2.5 mL of pre-cooled methyl butyl ether was added and 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, centrifugation was performed at 1000 g for 10 min. The upper organic phase (MTBE) was collected, and 1 mL of mixed solvent (methyl tert-butyl ether / methanol / water (10:3:2.5, v / v / v)) was added to the lower layer (water and methanol) and extracted again, and the upper organic phase was collected. The organic phases collected twice were concentrated by nitrogen blowing with a nitrogen blower. Redissolved with 100 μL of isopropanol, and then detected and analyzed by the LC-MS / MS system.

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

[0063] Example 4: The present invention also provides the use of the Fucha lipid vesicle-like nanoparticles in drugs for treating ulcerative colitis.

[0064] In order to illustrate the application of the Fucha lipid vesicle-like nanoparticles obtained by the present invention, the present invention provides the following experimental evidence:

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

[0066] Seven-week-old male C57BL / 6J mice were raised in the Experimental Animal Center of the Medical Research Institute of Northwestern Polytechnical University. After one week of free diet adaptation, five mice were randomly selected as the normal group, and the other experimental mice were randomly divided into two groups: DSS modeling group and Fucha lipid vesicle-like nanoparticle group.

[0067] On days 0-10, mice in the DSS modeling group were gavaged with normal saline, and mice in the Fu'er tea lipid vesicle-like nanoparticle group were gavaged with Fu'er tea lipid vesicle-like nanoparticles. On days 11-17, the inflammatory bowel disease mouse model was established by free drinking of 2.5% DSS: each mouse was marked. The status of the mice was checked every day, and the water bottle was checked for leakage.

[0068] 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 stool conditions.

[0069] The sum of the weight loss percentage, diarrhea score, and blood in stool score is used to calculate the disease activity index (DAI), which is the DAI value. That is, DAI = (body weight index + stool shape + bleeding).

[0070] DAI scoring criteria: Symptoms were scored once a day: weight loss percentage of 0-1% was 0 points, 1%-5% was 1 point, 5%-10% was 2 points, 10%-20% was 3 points, and ≥20% was 4 points; normal stool characteristics were scored as 0 points, loose stools were scored as 1 point, unformed stools were scored as 2 points, and watery stools were scored as 3 points; normal stools without blood were scored as 0 points, stools with blood streaks were scored as 1 point, blood streaked stools were scored as 2 points, and heavy bleeding was scored as 3 points.

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

[0072] Figure 7 Schematic diagram of the modeling of ulcerative colitis treated with Fu'er tea lipid vesicle-like nanoparticles. After seven days of adaptive feeding, the mice were gavaged with Fu'er tea lipid vesicle-like nanoparticles for ten days, and then the model was established again to evaluate its therapeutic efficacy.

[0073] Figure 8 This is a graph of weight changes in the treatment of ulcerative colitis with Fu'an tea lipid vesicle nanoparticles. During the entire treatment period, it shows that the weight loss in the treatment of ulcerative colitis with Fu'an tea lipid vesicle nanoparticles is about 3%, while the weight loss in the modeling group is 12%, indicating that the treatment of ulcerative colitis with Fu'an tea lipid vesicle nanoparticles can inhibit weight loss and improve the symptoms of weight loss.

[0074] Fig. 9 This is a scoring chart of the disease activity index of ulcerative colitis treated with Fu'an tea lipid vesicle-like nanoparticles. During the entire treatment period, it shows that the disease activity index of ulcerative colitis treated with Fu'an tea lipid vesicle-like nanoparticles was significantly lower than that of the modeling group, indicating that Fu'an tea lipid vesicle-like nanoparticles can improve the status of the entire disease process and reduce fecal bleeding and diarrhea.

[0075] Fig.10This is a graph of the colon length of ulcerative colitis treated with Fu'an tea lipid vesicle-like nanoparticles. The colon length represents the state of colon damage in ulcerative colitis mice. The longer the length, the better the effect of the treatment intervention. During the entire treatment period, the colon length of ulcerative colitis treated with Fu'an tea lipid vesicle-like nanoparticles was significantly longer than that of the modeling group. During the entire treatment period, the colon length of ulcerative colitis treated with Fu'an tea lipid vesicle-like nanoparticles was about 6.4 cm, while the modeling group decreased by about 5.5 cm, indicating that the treatment of ulcerative colitis with Fu'an tea lipid vesicle-like nanoparticles can improve the state of colon damage, reduce colon damage, and show excellent therapeutic effects.

[0076] Fig.11 The colon pathology of ulcerative colitis treated with Fu'er tea lipid vesicle-like nanoparticles was hematoxylin-eosin stained to detect and score the pathology of colon tissue. The intestinal epithelium and crypt structures in the colon tissue of the model group were severely damaged, and the structure almost disappeared. A large number of inflammatory cells were distributed under the intestinal mucosa, and there was extremely obvious swelling throughout the intestinal tissue. Fu'er tea lipid vesicle-like nanoparticles can protect intestinal epithelial cells and crypts, significantly reduce the inflammatory infiltration of colon tissue, and the damage to colon tissue has been significantly improved.

[0077] Fig.12 Goblet cells and the mucus they secrete are important barriers that prevent 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 synthesis and secretion of mucin, lead to intestinal mucosal barrier dysfunction. The expression of mucin in colon tissue was evaluated by Asley blue staining. The stain Asley blue can stain acidic mucin in blue. The results showed that the number of goblet cells and mucin in the model group was much less than that in the Fu'er tea lipid vesicle-like nanoparticle group. The results showed that Fu'er tea lipid vesicle-like nanoparticles can significantly improve the damage of goblet cells in colitis.

[0078] The above figures show that the Fu'an tea lipid vesicle-like nanoparticles provided by the present invention have the potential to prevent weight loss, and the DAI score of the experimental mice given Fu'an tea lipid vesicle-like nanoparticles by gavage is significantly lower than that of the model group. The experimental mice given Fu'an tea lipid vesicle-like nanoparticles by gavage significantly prevented the shortening of the colon. In summary, it shows that gavage of Fu'an tea lipid vesicle-like nanoparticles has a good anti-inflammatory effect, can well improve the weight loss and bloody stool during the course of ulcerative colitis, and provides an effective treatment for ulcerative colitis.

[0079] like Fig.13The safety of Fu'an tea lipid vesicle nanoparticles during oral administration was evaluated. After oral gavage for seven days, the heart, liver, spleen, lung and kidney of mice were taken for pathological staining. The section results showed that there was no obvious change in the morphology of various tissues and organs, indicating that the treatment did not produce obvious toxic side effects and had good biosafety. In summary, the oral administration of Fu'an tea lipid vesicle nanoparticles has a high in vivo safety and can be used as 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting and preparing Fucha lipid vesicle-like nanoparticles, characterized in that: The steps include: Mixing Fu tea and ultrapure water in a mass ratio of 1:6 to 1:10 to squeeze out the juice, and then passing through a 100-200 mesh filter to obtain Fu tea stock solution; Then, the Fu tea stock solution was subjected to gradient centrifugation to obtain a supernatant; Finally, the supernatant is centrifuged at a relative centrifugal force higher than the highest relative centrifugal force of the gradient centrifugation, and then a standard phosphate buffer having a mass ratio of 1:8 to 1:10 is added to resuspend the precipitate to obtain Fu'er tea lipid vesicle-like nanoparticles.

2. The method for preparing the Fu'er tea lipid vesicle-like nanoparticles according to claim 1, wherein the Fu'er tea is washed before being mixed with ultrapure water, and the specific washing process is: Pour the Fu tea into a container, wash it at least 3 times with purified water, then wash it at least 3 times with distilled water, then soak it in 6 to 10 times the distilled water for at least 30 minutes to ensure that the leaves are completely moistened.

3. The method for preparing Pu'er tea lipid vesicle-like nanoparticles as described in claim 1 is characterized in that the mixed juice of Pu'er tea and ultrapure water is carried out in a juicer with a juicer power of 200 to 1000 w and a juicer time of at least 2 min, so that the Pu'er tea is completely broken.

4. The method for preparing the Fu'er tea lipid vesicle-like nanoparticles according to claim 1, wherein the specific steps of subjecting the Fu'er tea stock solution to gradient centrifugation are: Pour the Pu'er tea stock solution into a centrifuge bottle, and centrifuge it at a relative centrifugal force of 1000-2000g for 10-20min, at a relative centrifugal force of 3000-6000g for 20-40min, and at a relative centrifugal force of 10000-12000g for 40-60min to obtain the supernatant to remove large particles and crude fiber in the Pu'er tea.

5. The method for preparing Pu'er tea lipid vesicle-like nanoparticles as described in claim 1, characterized in that the supernatant is centrifuged at a relative centrifugal force of 130,000 to 150,000 g and a centrifugal time of 60 to 70 min.

6. The method for preparing Fu'er tea lipid vesicle-like nanoparticles as claimed in claim 1, wherein the Fu'er tea is Fu'er tea or dark tea made from dark tea.

7. A kind of Pu'er tea lipid vesicle-like nanoparticles obtained by the preparation method as described in claims 1-6, characterized in that the Pu'er tea lipid vesicle-like nanoparticles have a particle size of 180-380 nm, a yield of 0.3-0.8 mg / g Pu'er tea, and appear disc-shaped or tray-shaped under a transmission electron microscope and a lipid membrane structure under a scanning electron microscope.

8. The Fu'an tea lipid vesicle-like nanoparticles according to claim 7, characterized in that: The lipid components in the Pu'er 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.

9. An application of the Fu'er tea lipid vesicle-like nanoparticles as claimed in claim 7, characterized in that: Application of the Pu'er tea lipid vesicle-like nanoparticles in medicine for treating ulcerative colitis.

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