Preparation method and application of berberin-vitamin D3 colon-targeted micelle

Abstract

The invention discloses a preparation method of a berberin-vitamin D3 colon-targeted micelle and application of the berberin-vitamin D3 colon-targeted micelle in preparation of a medicine for treating inflammatory enteritis. The berberin-vitamin D3 colon-targeted micelle can be released in a positioned mode under the colitis high-concentration ROS microenvironment condition; the bioavailability of berberine and vitamin D3 is improved, the side effects of the whole body are reduced, and targeted therapy of IBD is realized; in combination with in vivo and in vitro experiments, the berberin-vitamin D3 colon-targeted micelle systematically verifies the effectiveness of the berberin-vitamin D3 colon-targeted micelle in treatment of DSS-induced colitis, promotes clinical transformation of nanoparticles, and provides theoretical support for clinical prevention and treatment of IBD.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, and in particular to a method for preparing berberine-vitamin D3 colon-targeted micelles and application of the micelles in preparing drugs for treating inflammatory bowel inflammation. Background Art

[0002] Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD); it is a chronic digestive tract disease characterized by intestinal inflammation, diarrhea, and blood in the stool. IBD has a long course, is prone to recurrence, and is not cured, which seriously reduces the quality of life of patients.

[0003] The pathogenesis of IBD is complex and has not yet been fully elucidated. In addition to genetic and environmental factors, it is generally believed that it is closely related to intestinal barrier dysfunction caused by persistent intestinal inflammation, intestinal microbial imbalance, and intestinal mucosal immune imbalance. During the active phase of IBD, the generation of reactive oxygen species (ROS) in the intestinal mucosa increases significantly by 10 to 100 times, leading to lipid peroxidation and triggering a pro-inflammatory signaling cascade, which in turn leads to the continuous production of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interferon γ (INF-γ), interleukin 1β (IL-1β), IL-6 and IL-17. The release of these pro-inflammatory molecules interferes with the tissue homeostasis of the intestinal mucosa and triggers excessive immune responses of immune cells in the intestine, thereby further inducing intestinal damage, such as changes in the tight junction (TJ) structure, increased apoptosis and bacterial translocation. Enteric pathogens and their metabolites activate the intestinal immune system through pattern recognition receptors (PRRs), promote Th17 cell differentiation by activating the STAT3 pathway, further accelerate the inflammatory response, and ultimately lead to intestinal flora dysbiosis, immune homeostasis imbalance and barrier dysfunction in patients with ulcerative colitis. Due to long-term high levels of inflammation in the body, more than 70% of IBD patients suffer from vitamin D (VD) deficiency, and 10% to 30% of patients even develop osteoporosis. Studies have shown that the degree of Vitamin D deficiency is positively correlated with the disease activity score (DAI) and C-reactive protein level of IBD patients.

[0004] The current IBD treatment program aims to control inflammation and relieve clinical symptoms to improve the patient's quality of life. The first-line drugs for treating UC are aminosalicylic acids, and the first-line drugs for treating CD are glucocorticoids. They are difficult to achieve satisfactory results in clinical practice. Not only are they ineffective for moderate to severe patients, the recurrence rate is high, and they cannot effectively solve the problem of colon epithelium / mucosal repair in patients, and they may even cause serious side effects. Therefore, new safe and effective therapies for treating IBD need to be developed.

[0005] Studies have shown that the intestinal permeability of IBD patients is significantly increased, prompting intestinal immune cells and macrophages to utilize the epithelial permeability and retention effect (EPR, similar to the EPR effect of tumor blood vessels) to preferentially accumulate and selectively absorb large molecules or nanoparticles. Therefore, the new nanoparticle carrier drug delivery system has great advantages in the treatment of IBD. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a berberine-vitamin D3 colon-targeted micelle and its application in preparing a drug for treating inflammatory bowel disease. The present invention combines the specific chemical signal of abnormally high ROS in the colon inflammation microenvironment to prepare a berberine-vitamin D3 colon-targeted micelle, which can directly deliver berberine to the inflammatory colon lesion site, which can not only improve the bioavailability of the drug, enhance the therapeutic effect of the drug, but also reduce systemic side effects; that is, using the characteristics of significantly increased ROS in the colon inflammation microenvironment, the hydrophobic vitamin D3 and the hydrophilic berberine are connected by covalent bonds that respond to ROS degradation, and then further prepared into negatively charged nano-micelles, and a berberine nano-colon-targeted delivery system based on ROS response is constructed. The drug releases the drug under the action of highly reactive oxygen ROS in the colon inflammation site, thereby reducing colon inflammatory factors and promoting the recovery of colon microecology, thereby achieving the purpose of treating IBD, and the application in the treatment of inflammatory bowel disease has a good therapeutic effect.

[0007] The object of the present invention is achieved in that: A method for preparing berberine-vitamin D3 colon-targeted micelles comprises the following steps: Step 1: Preparation of berberine: The specific process is as follows: take a set amount of berberine hydrochloride and put it into a vacuum drying oven for a period of time, put the dried berberine hydrochloride into a flask, evacuate and fill with nitrogen, and react the reaction bottle in an oil pan for a period of time under nitrogen protection conditions. During this process, the color of the product changes from yellow to orange to red in sequence; the obtained reaction product is extracted through a silica gel column to obtain a pure product, and the product is red, that is, berberine, and berberine is subjected to nuclear magnetic resonance hydrogen spectrum; Step 2: Preparation of thioketal: first, concentrated sulfuric acid is added dropwise to saturated saline to prepare and collect HCl gas, then 3-mercaptopropionic acid solution and acetone solution are mixed in a set ratio and added to a flask, HCl gas is introduced and stirred to react for a period of time, and after the reaction, the reaction product is added to ice salt water to terminate the reaction to obtain a crude thioketal, and then the crude thioketal is washed with n-hexane and ice water for at least three times, and finally vacuum dried to obtain the final product thioketal, and the nuclear magnetic resonance hydrogen spectrum and mass spectrum of the thioketal connecting bond are characterized; Step 3: Preparation of compound 1: reacting the set amount of berberine characterized by mass spectrometry and K2CO3 in acetonitrile solution in step 1 at a set temperature for a period of time to obtain a mixed solution; then adding a certain amount of 2-bromoethanol to the mixed solution, then gradually heating the reaction temperature to the set temperature and reacting for a period of time; after the reaction, slowly lowering the temperature to 25°C, and evaporating the remaining CH3CN solution by rotary evaporation to obtain a crude compound 1; the crude compound 1 is purified by silica gel column to obtain a pure compound 1 as a yellow solid; Step 4: Preparation of Compound 2: Reflux the set compound 1 in step 3 in a CH3OH solution for a period of time, then gradually add a set amount of sodium borohydride to the solution in batches, and vigorously stir at a set temperature for 12 hours; after the reaction is completed, evaporate the remaining CH3OH solution by rotary evaporation to obtain a crude compound 2; purify the crude compound 2 by a silica gel column to obtain a pure compound 2 as a light yellow solid; Step 5: Preparation of compound 3: Dissolve the compound 2 in step 4 in a dichloromethane solution, then add it to a mixed system of DMAP, DCC, thioketal and dichloromethane. Under dry N2 conditions, react the mixed system at a set temperature for a period of time, and evaporate the remaining dichloromethane solution by rotary evaporation; then purify it by silica gel column to obtain pure compound 3 as a light yellow solid; Step 6: Preparation of compound 4: Dissolve vitamin D3 in a dichloromethane solution, and gradually add the dichloromethane solution of vitamin D3 to a dichloromethane mixture system of DCC, pure compound 3 in step 5, and DMAP under dry nitrogen, and react at a set temperature for a period of time, and evaporate the residual dichloromethane solution by rotary evaporation; purify by silica gel column to obtain pure compound 4, which is a light yellow solid; Step 7: Preparation of compound 5: Dissolve the compound 4 in step 6 in a chloroform solution, then gradually add the N-bromosuccinimide solution dropwise to the chloroform solution of compound 4, react the reaction system at a set temperature for a period of time, then lower the temperature of the reaction solution to 25°C, and filter; after filtration, purify the filtrate through a silica gel column to obtain pure compound 5, the product is a yellow solid; that is, pure compound 5 is a BBR-TK-VD3 conjugate; Step 8: Preparation of BBR-TK-VD3 nanomicelles: BBR-TK-VD3 and TPGS were mixed in different mass ratios to obtain three mixed solutions of mixed solution 1, mixed solution 2, and mixed solution 3. Different amounts of the three mixed solutions were respectively dropped into 3 mL of water. The mixture was gently shaken to obtain nanoparticles containing BBR-TK-VD3 and TPGS in different mass ratios. The particle size and potential of the nanoparticles were measured at the three ratios before and after passing through the water membrane using a particle size analyzer. The optimal condition was a mixed solution with a BBR-TK-VD3 and TPGS ratio of 5:1. 50 μL of the mixed solution was dropped into 3 mL of water.

[0008] In the step 1, berberine hydrochloride is placed in a 60° C. vacuum drying oven and dried for 2 h. The reaction bottle is reacted in an oil pan at 190° C. for 15 to 45 min under nitrogen protection. The volume ratio of pure ethanol to dichloromethane in the mobile phase of the silica gel column is 1:4 to 10.

[0009] In the step 2, the 3-mercaptopropionic acid solution and the acetone solution are mixed in a molar ratio of 2:1-2 and added to a flask, and HCl gas is introduced and stirred to react for 4 hours.

[0010] In the step 3, 1 mol of berberine and 1 mol of K2CO3 in acetonitrile solution in step 1 are reacted at 50°C for 30 minutes to obtain a mixed solution, 2 mol of 2-bromoethanol solution is added to the mixed solution, and then the reaction temperature is gradually heated to 82°C and continued for 24 hours.

[0011] In the step 4, 1 mol of compound 1 in step 3 is refluxed in a CH3OH solution for 15 min, and then 2-5 mol of solid sodium borohydride is gradually added to the solution in batches, and stirred at 0°C for 12 hours.

[0012] In the step 5, 1 mol of compound 2 in step 4 is dissolved in a dichloromethane solution, and then added to a mixed system of DMAP, DCC, thioacetal (molar ratio 0.8~1.5:1~1.5:0.2~0.5) and dichloromethane, and the mixed system is reacted at a temperature of 0°C for 48 hours under dry nitrogen conditions.

[0013] In the step 6, 1 mol of vitamin D3 solid is dissolved in a dichloromethane solution, and the dichloromethane solution containing vitamin D3 is gradually added to a dichloromethane mixture system containing DCC, pure compound 3, and DMAP (molar ratio of 0.8~2.5:1~1.5:0.2~0.5) under dry nitrogen conditions, and the reaction is carried out at a temperature of 0°C for 48 hours.

[0014] In step 7, 1 mol of compound 4 is dissolved in a chloroform solution, and then 1-2 mol of N-bromosuccinimide solution is gradually added dropwise to the chloroform solution containing compound 4. The reaction system is reacted at 50° C. for 6 h, and then the temperature of the reaction solution is lowered to room temperature.

[0015] In the step 8, the BBR-TK-VD3 solid and the TPGS solid are mixed in different proportions, 5 mg of the prepared BBR-TK-VD3 conjugate is weighed and dissolved in 1 mL of DMSO solution, and then 1 mg, 2 mg, and 5 mg of TPGS solid are added thereto respectively to obtain three mixed solutions 1, mixed solution 2, and mixed solution 3, and 50 μL, 100 μL, and 150 μL of the three mixed solutions are respectively dropped into 3 mL of water and gently shaken to obtain nanoparticles containing BBR-TK-VD3 and TPGS in different proportions, and the ratio of BBR-TK-VD3 to TPGS is 5:1, 5:2, and 1:1.

[0016] Application of berberine-vitamin D3 colon-targeted micelles in the preparation of drugs for treating inflammatory bowel inflammation.

[0017] The beneficial effects of the present invention are: (1) the berberine-vitamin D3 colon-targeted micelles of the present invention can achieve localized release under the conditions of high-concentration ROS microenvironment of colitis, thereby improving the bioavailability of berberine and vitamin D3 and reducing systemic side effects, thereby achieving targeted treatment of IBD; (2) The present invention combines in vitro and in vivo experiments to systematically verify the effectiveness of the berberine-vitamin D3 colon-targeted micelles in treating DSS-induced colitis, promote the clinical transformation of nanoparticles, and provide theoretical support for the clinical prevention and treatment of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a synthetic route diagram of the conjugate BBR-TK-VD3 of the present invention; Figure 2 This is the nuclear magnetic resonance characterization of the conjugate BBR-TK-VD3 in the present invention Figure 3 The UV absorption diagram of the conjugate BBR-TK-VD3 and important intermediates in the present invention; Figure 4 The particle size and Zeta potential diagram of BBR-TK-VD3 NPs in the present invention; Figure 5 The electron microscope image of BBR-TK-VD3 NPs and the particle size statistics of the present invention; Figure 6 The particle size and potential stability results of BBR-TK-VD3 NPs in the present invention after being placed at room temperature for 14 days (n=3); Figure 7 This is the TK degradation diagram of BBR-TK-VD3 NPs in the present invention in vitro in response to oxidation at different concentrations of H2O2; Figure 8 The BBR-TK-VD3 NPs of the present invention inhibit the NO secretion of Raw 264.7 macrophages induced by LPS; Fig. 9 The BBR-TK-VD3 NPs of the present invention inhibit the secretion of TNF-α by Raw 264.7 macrophages induced by LPS; Fig.10 The effect of BBR-TK-VD3 NPs on the viability of RAW264.7 cells in the present invention; Fig.11 A flow chart for establishing an inflammatory bowel disease (IBD) mouse animal model in the present invention; Fig.12 The results of the BTV NPs in the present invention in alleviating diarrhea in mice and reducing the disease activity index in mice; Fig.13 This is the result of the BTV NPs in the present invention improving the weight loss of IBD mouse models; Fig.14 The results of the present invention show that BTV NPs improve the reduction of colon length and swelling in the mouse IBD mouse model; Fig.15 The results of BTV NPs in the present invention reducing the spleen index of IBD model mice; Fig.16 The BTV NPs of the present invention inhibit the production of MPO in IBD model mice; Fig.17 The BTV NPs of the present invention inhibit the production of TNF-α (A) and IL-6 (B) in IBD model mice; Fig.18 This is the H&E staining image of the colon tissue of BTV NPs and each control group in the invention. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0020] Part 1 1. Preparation of BBR-TK-VD3 Nanomicelles 1.1 Synthesis of BBR-TK-VD3 conjugate The synthetic route of BBR-TK-VD3 conjugate is as follows Figure 1 As shown: Preparation of berberrubine: Take the berberine hydrochloride solid and put it into a 60℃ vacuum drying oven to dry for 2h, put the dried berberine hydrochloride into a flask, evacuate and fill with nitrogen, and react the reaction flask in a 190℃ oil pan for 15min under nitrogen protection. During this process, the color of the product changes from yellow to orange to red. The obtained reaction product is extracted through a silica gel column (column mobile phase: pure ethanol: dichloromethane = 1:4), the product is red, and berberrubine is subjected to nuclear magnetic resonance hydrogen spectrum.

[0021] Preparation of thioketal (TK): First, concentrated sulfuric acid was added dropwise to saturated brine to prepare and collect HCl gas, then 3-mercaptopropionic acid solution and acetone solution were mixed in a molar ratio of 2:1.5 and added to a flask, HCl gas was introduced and stirred for 4 hours, and after the reaction, the reaction product was added to ice brine to terminate the reaction to obtain crude TK, and then the crude TK was washed with n-hexane and ice water for at least three times, and finally vacuum dried to obtain the final product TK.

[0022] Preparation of compound 1: 1 mol of berberine characterized by mass spectrometry and 1 mol of K2CO3 in acetonitrile solution were reacted at 50 °C for 30 min to obtain a mixed solution. 2 mol of 2-bromoethanol was then added to the mixed solution, and the reaction temperature was gradually heated to 82 °C and continued for 24 h. After the reaction was completed, the temperature was slowly lowered to 25 °C, and the remaining CH3CN solution was evaporated by rotary evaporation to obtain a crude compound 1. The crude compound 1 was purified by silica gel column to obtain a yellow solid compound.

[0023] Preparation of compound 2: 1 mol of compound 1 was refluxed in a CH3OH solution for 15 min, and then a certain amount of sodium borohydride was gradually added to the solution in batches, and vigorously stirred at 0°C for 12 hours. After the reaction was completed, the remaining CH3OH solution was evaporated by rotary evaporation to obtain a crude compound 2. The crude compound 2 was purified by a silica gel column to obtain pure compound 2, and the product was a light yellow solid.

[0024] Preparation of compound 3: 1 mol of compound 2 was dissolved in a dichloromethane solution, and then added to a mixed system of DMAP, DCC, TK and dichloromethane. The mixed system was reacted at 0 °C for 48 h under dry N2 conditions, and the remaining dichloromethane solution was evaporated by rotary evaporation. Then, it was purified by silica gel column to obtain pure compound 3, and the product was a light yellow solid.

[0025] Preparation of compound 4: 1 mol of vitamin D3 was dissolved in 20 mL of dichloromethane solution, and the dichloromethane solution of vitamin D3 was gradually added to the dichloromethane mixture system of DCC, compound 3, and DMAP under dry N2 conditions, and reacted at 0°C for 48 h, and the residual dichloromethane solution was evaporated by rotary evaporation. Pure compound 4 was obtained by purification on a silica gel column, and the product was a light yellow solid.

[0026] Preparation of compound 5: 1 mol of compound 4 was dissolved in 10 mL of chloroform solution, and then N-bromosuccinimide (NBS) solution was gradually added dropwise to the chloroform solution of compound 4. The reaction system was reacted at 50 °C for 6 h, then the temperature of the reaction solution was lowered to room temperature and filtered. After filtration, the filtrate was purified by silica gel column to obtain pure compound 5 as a yellow solid.

[0027] 1.2 Preparation and characterization of BBR-TK-VD3 nanomicelles 1.2.1 Preparation of BBR-TK-VD3 nanomicelles Preparation of nanoparticles by dropwise addition method and screening of the mixing ratio of BBR-TK-VD3 and TPGS: BBR-TK-VD3 and TPGS were mixed in different mass ratios (BBR-TK-VD3: TPGS = 5:1, 5:2, 1:1), that is, 5 mg of three prepared BBR-TK-VD3 conjugates were weighed and dissolved in three 1 mL DMSO solutions, and then 1, 2, and 5 mg TPGS were added thereto respectively to obtain three mixed solutions 1, 2, and 3. 50, 100, and 150 μL of the three mixed solutions were respectively dropped into 3 mL of water, and the mixture was gently shaken to obtain nanoparticles containing different ratios of BBR-TK-VD3 and TPGS. The particle size and potential of the nanoparticles were measured at the three ratios before and after passing through the water membrane using a particle size analyzer. The optimal condition was a mixed solution with a BBR-TK-VD3 and TPGS ratio of 5:1, and 50 μL of the mixed solution was dropped into 3 mL of water.

[0028] 1.2.1 Characterization of BBR-TK-VD3 nanomicelles Morphology of nanomicelles: The morphology of BBR-TK-VD3 nanomicelles was observed using scanning electron microscopy and transmission electron microscopy.

[0029] Stability of nanomicelles: 50 μL of a mixed solution of BBR-TK-VD3 and TPGS in a mass ratio of 5:1 was dropped into 3 mL of water to prepare nanomicelles. The solution was placed at room temperature for 14 days, and the particle size and potential of the nanomicelles were measured at 0, 2, 4, 7, and 14 days, respectively.

[0030] Encapsulation efficiency of nanomicelles: To determine the drug encapsulation efficiency of BBR-TK-VD3 nanomicelles, 50 μL of a mixed solution of BBR-TK-VD3 and TPGS in a mass ratio of 5:1 was dropped into 3 mL of water to prepare nanomicelles. The nanomicelles were dialyzed for 6 h, and the drug concentration of the solution before and after dialysis was measured by ultraviolet. The encapsulation efficiency was calculated according to the following formula; Encapsulation efficiency = (1- C f / C t )×100% Note; Cf is the free drug concentration; Ct is the total drug concentration in the suspension.

[0031] Study on the H2O2 oxidative stimulation response of nanomicelles: After preparation of BTV nanomicelles, they were passed through a membrane and dialyzed for 6 hours. Oxidative stimulation was performed with different concentrations of hydrogen peroxide: 100μM, 250μM, 500μM, 2.5mM, 5mM, 12.5mM, 25mM, 50mM, 100mM, and 200mM, and the absorbance changes were recorded at 218nm within 5 min-8 h.

[0032] Part 2 1 Experimental cells and animals 60 SPF KM mice, 7-8 weeks old, male, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., animal license number SCXK (Lu) 2019003, experimental unit use license number SYXK (Yu) 200-0004. They were kept in the applicant's experimental animal room.

[0033] 2Drugs, reagents and instruments 2.1 Drugs and reagents Reagent name Manufacturer Berberine Hydrochloride Shanghai MacLean Biochemical Co., Ltd. 3-Mercaptopropionic acid Shanghai Aladdin Biochemical Technology Co., Ltd. Vitamin E polyethylene glycol succinate (TPGS) Shanghai Aladdin Biochemical Technology Co., Ltd. Dichloromethane Tianjin Fuyu Essence Co., Ltd. Pure ethanol Tianjin Fuyu Essence Co., Ltd. Acetonitrile Honeywell 2-Bromoethanol Shanghai MacLean Biochemical Co., Ltd. Potassium carbonate Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd. 4-Dimethylaminopyridine (DAMP) Shanghai Aladdin Biochemical Technology Co., Ltd. Dicyclohexylcarbodiimide (DCC) Shanghai Aladdin Biochemical Technology Co., Ltd. N-Bromosuccinimide (NBS) Shanghai Aladdin Biochemical Technology Co., Ltd. DMEM Solebow Technology Co., Ltd. Fetal bovine serum GIBCO (Australia) <![CDATA[Vitamin D3]]> Shanghai Aladdin Biochemical Technology Co., Ltd. Mesalazine Enteric-coated Tablets Sunflower Pharmaceutical Group Paraformaldehyde McLean Biochemical Technology Co., Ltd. Mouse IL-1β, IL-6, TNF-α, etc. ELISA kits BD BiosciencesTM 2.2 Instruments Instrument Name Manufacturer Electronic precision balance Mettler-Toledo Instruments (Shanghai) Co., Ltd. Digital constant temperature electric heating jacket Tianjin Sadelis Laboratory Analytical Instruments Factory Vacuum drying oven Linhai Tanshi Vacuum Equipment Co., Ltd. Oil bath pot Shanghai Ailang Instrument Co., Ltd. NanoBrook90PlusPALS Particle Size Analyzer Brookhaven Instruments Rotary evaporator Shanghai Ailang Instrument Co., Ltd. Low temperature high speed centrifuge Hunan Xiangyi Experimental Instrument Development Co., Ltd. Vacuum freeze dryer Shanghai Ailang Instrument Co., Ltd. UV spectrophotometer Thermo Fisher Scientific High performance liquid chromatography Waters Technologies Inc. Ultrasonic cleaning machine Ningbo Xinzhi Biotechnology Co., Ltd. Plate washer TECAN Multifunctional ELISA reader (Filter Max F5)Molecular Devices Tissue grinding instruments JieLing Instrument Manufacturing (Tianjin) Co., Ltd. Decolorization shaker Changzhou National Laboratory Equipment Institute 3% DSS modeling solution: Dissolve 3 g of dextran sulfate sodium in 100 mL of water to obtain a 3% DSS solution.

[0034] Mesalazine solution: According to the drug instructions, in the case of acute attack, the adult dose is 1g (4 tablets) once, 4 times a day. According to the body surface area conversion method, the equivalent dose ratio of mice and humans is 9.1, and mesalazine is given at 200 mg / kg.

[0035] Berberine group: According to the drug instructions and the body surface area conversion method, berberine was given at 50 mg / kg and berberine was prepared into a 50 mg / mL solution.

[0036] Drug administration group (BTV): The BBR-TK-VD3 nanomicelle solution was prepared into 50 mg / mL, and the BTV administration dose was the same as the berberine equivalent, which was also 50 mg / kg.

[0037] 3 Methods 3.1 Cell experiments 3.1.1 Cytotoxicity assay Raw 264.7 rat macrophages were inoculated in 24-well plates. After the cells adhered to the wall, they were treated with different concentrations of berberine, VD3, berberine + VD3, and BBR-TK-VD3 micelles (0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 10, and 200 μmol / L) for 24 hours. Then, 20 μL MTT (5 mg / mL) was added to each well. The culture was continued for 4 hours, the culture medium was discarded, and 100 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve. The absorbance (A value) was detected at 570 nm and 450 nm as the reference wavelength. The cell growth survival rate was calculated according to the following formula: Cell growth survival rate (%) = (OD570 给药 -OD450 给药 ) / (OD570 空白 -OD450 空白 ).

[0038] 3.1.2 Anti-inflammatory experiments Rat macrophage Raw 264.7 cells were seeded in 24-well plates, and after the cells adhered, they were treated with berberine, VD3, berberine + VD3, and BBR-TK-VD3 micelles in the presence or absence of LPS (100 ng / ml) for 24 hours. The culture supernatant was collected and the TNF-α factor and NO concentration were determined by ELISA.

[0039] 3.2 Animal experiments 3.2.1 Model preparation and drug administration like Figure 1 As shown in the figure, male Kunming mice were used as experimental animals, DSS was used as modeling drug, and free drinking of DSS solution was adopted for modeling. Figure 2 shown.

[0040] Acute colitis modeling method: After one week of adaptive feeding, male Kunming mice were allowed to drink 3% DSS solution freely every day from 1 to 7 days. During this period, a certain amount of drugs were taken orally (gavage) at the same time. On the 8th day, they were killed to collect key organs, and the efficacy of drug treatment was evaluated by analyzing histological and evaluation index results.

[0041] (1) Acute modeling stage: Except for the blank group, the mice in other groups drank 3% DSS solution freely, while the mice in the blank group drank pure water.

[0042] (2) Drug administration phase (oral gavage): Normal group (Naive): pure water; Model group (Model): pure water; Mesalazine group (5-ASA): mesalazine solution; Berberine group (BBR): berberine solution; Drug-treated group (BTV): BBR-TK-VD3 micelle solution.

[0043] 3.2.2 Evaluation of body weight, physical signs and indicators The mice were weighed every day, and their rectal temperatures were measured and feces were recorded every two days.

[0044] 0: no weight loss, no temperature change, no diarrhea, no blood; 1: 1-3% weight loss, 1-2℃ drop in temperature, mild diarrhea; 2: 3-6% weight loss, 2-3℃ drop in temperature, loose stools, blood in stool; 3: 6-9% weight loss, 3-4℃ drop in temperature, diarrhea; 4: >9% weight loss, >4℃ drop in temperature, greater than diarrhea, heavy bleeding.

[0045] The mice were weighed before being killed, and the spleen and colon were taken and weighed after being killed. The length of the colon of each mouse was measured, and the spleen organ index and colon swelling rate were calculated.

[0046] 3.2.3 Cytokine detection ELISA kits were used to determine the concentration changes of MPO in mouse colon tissue and inflammatory factors IL-1β, IL-6 and TNF-α in colon tissue. The weighed mouse colon tissue was thawed on ice. According to the instructions of the kit, the colon tissue was mixed with pre-cooled PBS buffer (pH=7.4) at a weight-to-volume ratio (w / v) of 1:9 using a high-speed tissue homogenizer. The colon tissue was then centrifuged (4°C; 4000 rpm / min; 20 min) to obtain the supernatant. Subsequent determination steps were strictly carried out in accordance with the instructions of the ELISA kit.

[0047] 3.2.4 H&E staining to observe pathological changes of lung tissue The mice were killed, and the colon tissues were obtained and fixed in 4% paraformaldehyde, and the specimens were embedded in paraffin and sliced. The pathological specimens were stained with hematoxylin-eosin (H&E). The pathological changes of inflammatory infiltration, tissue damage, and intestinal crypt loss in various tissues were observed.

[0048] 4. Statistical processing SPSS 19.0 software was used for statistical analysis. All data were expressed as mean ± SD. One-way ANOVA was used for comparison among multiple groups, and t-test was used for comparison between two groups. p The difference was statistically significant when the value was <0.05.

[0049] result 1. Preparation and characterization of BBR-TK-VD3 nanomicelles Conjugate synthesis: Identified by NMR, UV and mass spectrometry, the results are as follows Figure 3 and Figure 4 As shown, the 1H NMR results and the UV results of each intermediate showed that the final product BBR-TK-VD3 conjugate was successfully synthesized.

[0050] Compound 1: 1H NMR (500 MHz, DMSO-d) δ 9.09 (s, 1H), 7.99 (s, 1H), 7.61(s, 1H), 7.22 (s, 1H), 6.96 (s, 1H), 6.38 (d, J = 7.8 Hz, 1H), 6.10 (s, 2H),4.49 (t, J = 6.1 Hz, 2H), 3.73 (s, 3H), 3.03 (d, J = 6.1 Hz, 2H), 1.06 (t, J= 7.0 Hz, 1H). Compound 2: 1H NMR (600 MHz, Chloroform- d ) δ 10.75 (s, 1H), 8.14 (s, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.32 (s, 1H), 6.83 (s,1H), 6.10 (s, 2H), 4.63 (s, 2H), 4.10 (s, 2H), 4.06 (s, 3H), 3.41 (s, 2H), 3.32 (s, 2H). Compound 3: 1H NMR (600 MHz, Chloroform-d) δ 6.90 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.73 (s, 1H), 6.59 (s, 1H), 5.92 (s, 2H), 4.27 (d,J = 15.6 Hz, 1H), 4.18 – 4.12 (m, 1H), 4.04 (ddd, J = 10.5, 5.6, 3.0 Hz, 1H), 3.86 (overlap, 5H), 3.60 – 3.49 (overlap, 2H), 3.24 (dd, J = 16.0, 3.8 Hz,1H), 3.18 (dt, J = 10.9, 2.9 Hz, 1H), 3.15 – 3.08 (m, 1H), 2.82 (dd, J =15.8, 11.4 Hz, 1H), 2.70 – 2.59 (overlap, 2H). Compound 5 (BBR-TK-VD3): 1H NMR (600 MHz, DMSO-d) δ 8.33 – 8.11 (m, 1H), 6.97 (t,1H), 5.58 (d, 2H), 2.57 (s, 8H), 1.96 (d, 1H), 1.71 (d, 10H), 1.65 –1.58 (m, 7H), 1.56 (s, 1H), 1.54 – 1.44 (m, 5H), 1.41 (d, 2H), 1.30 – 1.19(m, 12H), 1.17 – 1.09 (m, 6H), 1.04 (td,8H), 0.91 (s, 1H), 0.85 (s, 5H). Nanomicelle stability: Figure 5 As shown in the figure, the best condition was a mixed solution of BBR-TK-VD3 and TPGS with a ratio of 5:1. 50 μL of the mixed solution was dropped into 3 mL of water. Under this condition, the particle size, potential and dispersion of the nanomicelles formed before and after passing through the water membrane were the smallest. The hydrated particle size was about 77.79 ± 0.29 nm, the PDI was 0.206, and the potential was about -16.80 ± 1.43 mV. The TEM electron microscope image of BTV NPs showed that the particle size was mainly distributed in the range of 67.5 ± 5 nm ( Figure 6 ), which is consistent with the particle size measured by the nanoparticle size analyzer. Most of the nanoparticles are spherical and uniform in size, ranging from 62.5-77.5 nm, and the particle size distribution of nanomicelles is narrow. Figure 7 As shown, the freshly prepared nanomicelles were placed at room temperature for 14 days, and the particle size and Zeta potential of the nanomicelles were measured at 0, 2, 4, 7, and 14 days, respectively. The results showed that the nanomicelles were relatively stable within 14 days of storage at room temperature, and the particle size and potential were slightly reduced, but no obvious changes were shown.

[0051] Nanomicelle encapsulation efficiency: 50 μL of a mixed solution of BBR-TK-VD3 and TPGS in a ratio of 5:1 was dropped into 3 mL of water to prepare nanomicelles. The nanomicelles were dialyzed for 6 h, and the drug concentration of the solution before and after dialysis was measured by ultraviolet. The drug encapsulation efficiency was calculated to be 70.65%.

[0052] In vitro release of H2O2 in response to oxidative stimulation: Figure 4 From the UV graph of the intermediate, we can see that when the ROS-responsive TK linker is added, the UV increases the high absorption at around 218 nm, so we calculate the TK bond breakage by recording the absorbance at 218 nm. After preparing the BTV nanomicelles, DMSO was removed by dialyzing with a dialysis bag for 6 hours, filtered with a 200μm filter membrane, and oxidative stimulation was performed with different concentrations of hydrogen peroxide (100μM, 250μM, 500μM, 2.5mM, 5mM, 12.5mM, 25mM, and 50mM), and the absorbance was recorded at 218nm within 5min-8h. The results are shown in Figure 8 As shown in the figure, the in vitro drug release amount depends on the hydrogen peroxide concentration. The higher the hydrogen peroxide concentration, the more drug is released and the shorter the release time. When the hydrogen peroxide concentration is 50 mM, the release amount of BTV nanomicelles reaches 82.79%.

[0053] 2. Cell experiments The LPS-stimulated Raw264.7 macrophage inflammation model is widely used in the screening of anti-inflammatory active ingredients and the study of their mechanism of action. Macrophages are important effector cells in the inflammatory response process. They can activate immune responses through antigen recognition and antigen presentation. The recognition of a variety of inflammatory cytokines and other immune-related substances will further trigger signal cascades, inducing the expression of a variety of pro-inflammatory mediators and cytokines, such as TNF-α, IL-1β and IL-6, and reactive nitrogen free radicals such as NO will also be generated in large quantities. These pro-inflammatory mediators can destroy the tight junctions between epithelial cells, thereby increasing the permeability of the intestinal mucosa and directly acting on the intestinal mucosa to damage the intestinal mucosal epithelium. The overexpression and release of these inflammatory mediators are closely related to the development of inflammation. Therefore, reducing the secretion of macrophage inflammatory mediators is an important reference indicator for judging the anti-inflammatory effect of drugs.

[0054] The results of cell experiments are as follows Fig. 9As shown in the figure, at the same molar concentration level, there was no significant difference in the cell activity between the drugs in each group. Compared with the blank control group, the levels of NO and TNF-α in RAW 264.7 cells were significantly increased after being treated with 200 ng / mL LPS ( p < 0.01), compared with the LPS control group, BTV nanomicelles with molar concentrations of 3.125, 6.25, 12.5, 25, and 50 μmol / L could significantly inhibit the release of NO and TNF-α, with a certain dose-effect relationship, and at the same low molar concentration of 3.125, 6.25, and 12.5 μmol / L, the inhibitory effect of BTV nanomicelles was stronger, which was significantly different from that of berberine and berberine-VD3 mixed groups.

[0055] like Fig.10 and Fig.11 As shown in the results, the inhibition rates of NO release by BTV nanomicelles in the concentration range of 3.125 ~ 50 μmol / L were 53.60%, 54.05%, 64.41%, 69.59% and 72.64%, respectively. When the drug molar concentration was 3.125 μmol / L, the inhibitory effects of BTV nanomicelles on NO were 2.43 and 2.42 times higher than those of berberine and berberine-VD3 mixed groups, respectively. The inhibition rates of TNF-α release by BTV nanomicelles in the concentration range of 3.125 ~ 50 μmol / L were 53.82%, 58.96%, 62.47%, 62.80% and 64.67%, respectively. When the drug molar concentration was 3.125 μmol / L, the inhibitory effects of BTV nanomicelles on TNF-α were 9.98% and 5.34% higher than those of berberine and berberine-VD3 mixed groups, respectively. Based on the above results, it is speculated that the in vivo anti-inflammatory effect of BTV nanomicelles will be more significant, which can inhibit the increase in intestinal mucosal permeability caused by colon inflammation and better reduce intestinal mucosal epithelial damage.

[0056] 3. Animal Experiments The apparent severity of inflammatory bowel disease is mainly determined based on body weight, mental state, movements and behaviors, hair gloss, stool shape, etc. The results of drug treatment will be comprehensively judged according to the inflammatory bowel disease index (DAI) scoring standard and combined with multiple dimensions such as colon tissue pathological changes.

[0057] 3.1 BTV nanomicelles reduced weight loss and significantly decreased DAI scores in DSS-induced IBD mice After administration of 3% DSS, mice began to show symptoms such as slow movements, prone immobility, poor hair luster, and loose feces. The weight of mice in all experimental groups decreased to varying degrees ( Fig.12 ), mice developed diarrhea symptoms ( Fig.13), indicating that the mouse model was successfully established. Compared with the model group, the weight loss of mice in the nano-micelle group and the positive group (5-SAS) was alleviated. After 7 days of the experiment, the weight of mice in the model group decreased significantly, and the weight dropped to 79.65% of the initial weight. ( p <0.001), while the body weights of mice in the BBR, 5-SAS and BTV groups decreased to 81.95%, 88.16% and 85.70% of their initial body weights, respectively. P <0.05), mesalazine group ( p <0.001), nanomicelle group ( p <0.001) mice had significant weight loss compared with the model group mice. Fig.12 shown.

[0058] Except for the blank control group, the DAI scores of mice in all DSS model experimental groups increased significantly. At the end of the experiment, the DAI comprehensive score of mice in the model group was 2.50. Compared with the model group, the weight loss of mice in the BBR, 5-SAS and BTV treatment groups was delayed, and the stool consistency and fecal occult blood phenomenon were also improved to a certain extent. Their DAI scores were 2.20, 1.88 and 1.91, respectively. The DAI score of mice in the BTV nano-micelle group was significantly lower than that in the model group, and there was a statistical difference ( p <0.01), which is equivalent to mesalazine, such as Fig.13 As shown, but the dose is only 1 / 4 of mesalamine.

[0059] 3.2 BTV nanomicelles alleviated the reduction in colon length and increase in spleen index in model mice Depend on Fig.14 A shows that compared with the blank group, the colon length of mice in the model group was significantly reduced ( p < 0.01,14B); Due to the severe colon edema and inflammation in the model group mice, the swelling of their colon was also significantly increased ( p <0.001,14C). Compared with the model group, BBR ( p < 0.05)、5-SAS( p < 0.05) and BTV( p < 0.001) groups can alleviate the shortening of the mouse colon to varying degrees, and the colon lengths were 8.17cm, 8.96cm, and 9.17cm, respectively. The colon length of the BTV nano-micelle group was significantly different from that of the berberine group ( p < 0.001), while there was no significant difference between the BTV nanomicelle group and the mesalazine group.

[0060] The spleen is an important immune organ. As inflammation occurs and worsens, the spleen will swell ( Fig.15 ), during the experiment, the spleen coefficient of the model group mice increased significantly ( p <0.01, compared with the negative control group); compared with the spleen coefficient of mice in the model group, the spleen coefficient of the nanomicelle treatment group was significantly reduced ( p <0.01), nanomicelles can alleviate spleen inflammation to some extent.

[0061] 3.3 Biochemical indicators The results of IL-6 and TNF-α inflammatory factors detection are as follows Fig.16 As shown, compared with the negative control group mice, the concentrations of IL-6 and TNF-α cytokines in the colon of the model group mice were significantly increased ( p <0.01); Mesalazine can significantly reduce the levels of various inflammatory cytokines, and the levels of IL-6 and TNF-α cytokines decreased by 47.3% and 41.0%, respectively. Compared with the model group, the berberine group reduced the levels of IL-6 and TNF-α cytokines by an average of 47.1% and 29.0%, respectively, and the BTV nano-micelle group reduced the levels of IL-6 and TNF-α cytokines by an average of 62.8% and 43.6%, respectively. The results showed that the anti-inflammatory effect of the BTV nano-micelle group was better than that of berberine and mesalazine tablets alone.

[0062] When DSS induces colitis in mice, myeloperoxidase (MPO) can accelerate the infiltration of neutrophils. When MPO enters the extracellular fluid, it causes an increase in the concentration of MPO in the colon tissue, leading to the occurrence of colon inflammation. Studies have shown that the oxidative damage of the mucosal barrier can be repaired by inhibiting the production of MPO, thereby reducing the oxidative stress response in the colon tissue of colitis mice. In this study, the concentration of MPO in the mouse colon homogenate was measured ( Fig.17 ), the results showed that the MPO activity of the colon of the model group mice increased significantly (p < 0.001) to 6.59 (U / g), while the MPO activity of the BBR (p < 0.05), 5-SAS (p < 0.01) and BTV nano-micelle (p < 0.001) administration groups decreased to 5.82, 5.00 and 4.70 U / g, respectively, compared with the model group, which was consistent with the results of inhibiting NO production in vitro. In summary, DSS induced an increase in the concentration of MPO in colon tissue, and BTV nano-micelles can significantly reduce the production of MPO, thereby alleviating the oxidative stress response caused by DSS and repairing colon tissue damage.

[0063] 5.4 Pathological sections Pathological sections can intuitively evaluate the pathological changes of IBD model mice. Fig.18 These are H&E pathological sections of colon tissues of mice in each group at the end of treatment.

[0064] The results of H&E staining sections of colon tissue showed that the colon tissue structure of mice in the blank control group was clear, and goblet cells with clear structure and large number could be seen. For mice in the model group, due to the induction of DSS, there were fewer goblet cells in the colon tissue and the crypt structure was greatly damaged. BTV nano-micelles could increase the number of goblet cells and repair certain crypt structures.

Claims

1. A method for preparing berberine-vitamin D3 colon-targeted micelles, characterized in that: The following steps are involved: Step 1, preparation of berberine: The specific process is as follows: take a set amount of berberine hydrochloride and put it into a vacuum drying oven for a period of time, put the dried berberine hydrochloride into a flask, evacuate and fill with nitrogen, and react the reaction flask in an oil pan for a period of time under nitrogen protection conditions. During this process, the color of the product changes from yellow to orange to red in sequence; the obtained reaction product is extracted through a silica gel column to obtain a pure product, and the product is red, which is berberine; Step 2, preparation of thioketal: first, concentrated sulfuric acid is added dropwise to saturated saline to prepare and collect HCl gas, then 3-mercaptopropionic acid solution and acetone solution are mixed in a set ratio and added to a flask, HCl gas is introduced and stirred to react for a period of time, after the reaction, the reaction product is added to ice salt water to terminate the reaction to obtain a crude thioketal, and then the crude thioketal is washed with n-hexane and ice water for at least three times, and finally vacuum dried to obtain the final product thioketal; Step 3, preparation of compound 1: react a certain amount of berberine characterized by mass spectrum and acetonitrile solution of K2CO3 in step 1 at a set temperature for a period of time to obtain a mixed solution; then add a set amount of 2-bromoethanol to the mixed solution, and then gradually heat the reaction temperature to the set temperature and react for a period of time; after the reaction is completed, slowly reduce the temperature to 25°C, and evaporate the remaining CH3CN solution by rotary evaporation to obtain a crude compound 1; purify the crude compound 1 by silica gel column, and the product is a pure compound 1 in the form of a yellow solid; Step 4, preparation of compound 2: reflux the set compound 1 in step 3 in a CH3OH solution for a period of time, then gradually add a certain amount of sodium borohydride to the solution in batches, and vigorously stir at a certain temperature for 12 hours; after the reaction is completed, evaporate the remaining CH3OH solution by rotary evaporation to obtain a crude compound 2; purify the crude compound 2 by a silica gel column to obtain a pure compound 2 as a light yellow solid; Step 5, preparation of compound 3: dissolve the compound 2 in step 4 in a dichloromethane solution, add it to a mixed system of DMAP, DCC, thioketal and dichloromethane, react the mixed system at a set temperature for a period of time under dry N2 conditions, and evaporate the remaining dichloromethane solution by rotary evaporation; then purify it by silica gel column to obtain a pure compound 3 as a light yellow solid; Step 6, preparation of compound 4: dissolve vitamin D3 in a dichloromethane solution, and gradually add the dichloromethane solution of vitamin D3 to a dichloromethane mixture system of DCC, pure compound 3 in step 5, and DMAP under dry nitrogen, and react at a set temperature for a period of time, and evaporate the residual dichloromethane solution by rotary evaporation; purify by silica gel column to obtain pure compound 4, which is a light yellow solid; Step 7, preparation of compound 5: dissolve compound 4 in step 6 in chloroform solution, then gradually add N-bromosuccinimide solution dropwise to the chloroform solution of compound 4, react the reaction system at a set temperature for a period of time, then lower the temperature of the reaction solution to 25°C, and filter; after filtration, purify the filtrate through a silica gel column to obtain pure compound 5, the product is a yellow solid; that is, pure compound 5 is a BBR-TK-VD3 conjugate; Step 8. Preparation of BBR-TK-VD3 nanomicelles: BBR-TK-VD3 and TPGS were mixed in different mass ratios to obtain three mixed solutions, namely, mixed solution 1, mixed solution 2, and mixed solution 3. Different amounts of the three mixed solutions were dropped into 3 mL of water, and the mixture was gently shaken to obtain nanoparticles containing BBR-TK-VD3 and TPGS in different mass ratios. The particle size and potential of the nanoparticles were measured at the three ratios before and after passing through the water membrane using a particle size analyzer.

2. The method for preparing berberine-vitamin D3 colon targeting micelles according to claim 1, characterized in that: In the step 1, berberine hydrochloride is placed in a 60° C. vacuum drying oven and dried for 2 h. The reaction bottle is reacted in an oil pan at 190° C. for 15 to 45 min under nitrogen protection. The volume ratio of pure ethanol to dichloromethane in the mobile phase of the silica gel column is 1:4 to 10.

3. The method for preparing berberine-vitamin D3 colon targeting micelles according to claim 1, characterized in that: In the step 2, the 3-mercaptopropionic acid solution and the acetone solution are mixed in a molar ratio of 2:1-2 and added to a flask, and HCl gas is introduced and stirred to react for 4 hours.

4. The method for preparing berberine-vitamin D3 colon targeting micelles according to claim 1, characterized in that: In the step 3, 1 mol of berberine and 1 mol of K2CO3 in acetonitrile solution in step 1 are reacted at 50°C for 30 min to obtain a mixed solution, 2-4 mol of 2-bromoethanol solution is added to the mixed solution, and then the reaction temperature is gradually heated to about 82°C and continued for 24-48 hours.

5. The method for preparing berberine-vitamin D3 colon targeting micelles according to claim 1, characterized in that: In the step 4, 1 mol of compound 1 in step 3 is refluxed in a CH3OH solution for 15 min, and then 2 to 6 mol of solid sodium borohydride is gradually added to the solution in batches, and the mixture is stirred at 0°C for 12 hours.

6. The method for preparing berberine-vitamin D3 colon-targeted micelles according to claim 1, characterized in that: In the step 5, 1 mol of compound 2 in step 4 is dissolved in a dichloromethane solution, and then added to a mixed system of dichloromethane containing DMAP, DCC, and thioketal. Under dry nitrogen conditions, the mixed system is reacted at a temperature of 0° C. for at least 48 hours, and the molar ratio of DMAP, DCC, and thioketal is 0.2~0.4:1~2:0.8~1.

5.

7. The method for preparing berberine-vitamin D3 colon-targeted micelles according to claim 1, characterized in that: In the step 6, 1 mol of vitamin D3 solid is dissolved in 20 mL of dichloromethane solution, and the dichloromethane solution containing vitamin D3 is gradually added to a dichloromethane mixture system containing DCC, pure compound 3, and DMAP under dry nitrogen conditions, and reacted at 0°C for 48 h; the molar ratio of DCC, pure compound 3, and DMAP is 0.8~2.5:1~1.5:0.2~0.

5.

8. The method for preparing berberine-vitamin D3 colon-targeted micelles according to claim 1, characterized in that: In step 7, 1 mol of compound 4 is dissolved in a chloroform solution, and then 1-2 mol of N-bromosuccinimide solution is gradually added dropwise to the chloroform solution containing compound 4. The reaction system is reacted at 50° C. for 6 h, and then the temperature of the reaction solution is reduced to 25° C.

9. The method for preparing berberine-vitamin D3 colon-targeted micelles according to claim 1, characterized in that: In the step 8, the BBR-TK-VD3 solid and the TPGS solid are mixed in different mass ratios, 5 mg of the prepared BBR-TK-VD3 conjugate is weighed and dissolved in 1 mL of DMSO solution, and then 1 mg, 2 mg, and 5 mg of TPGS solid are added thereto respectively to obtain three mixed solutions 1, mixed solution 2, and mixed solution 3, 50 μL, 100 μL, and 150 μL of the three mixed solutions are respectively dropped into 3 mL of water, and gently shaken to obtain nanoparticles containing BBR-TK-VD3 and TPGS in different mass ratios, and the ratios of BBR-TK-VD3 to TPGS are 5:1, 5:2, and 1:

1.

10. Use of berberine-vitamin D3 colon-targeted micelles prepared by the preparation method according to any one of claims 1 to 9, characterized in that: It is used in the preparation of drugs for treating inflammatory bowel inflammation.

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