Folic acid-chicoric acid liposome for treating ulcerative colitis and preparation and application thereof

Abstract

The application discloses a folate-chicoric acid liposome for treating ulcerative colitis and a preparation and application thereof, and relates to the technical field of veterinary drugs. The folate-chicoric acid liposome is composed of egg yolk lecithin, cholesterol, chicoric acid and folate (mass ratio of 10-14:2-5:1-2:1-2), has targeting property, has the effect of targeting macrophages, inhibits the polarization of macrophages to M1 type, can improve the clinical symptoms (weight loss, hematochezia and the like) of ulcerative colitis mice, increase the colon length, reduce the DAI score, the CMDI score and the colon index, relieve the pathological damage of the colon and relieve intestinal inflammation. The liposome has the effect of treating ulcerative colitis mice, and can provide an efficient drug for treating ulcerative colitis in the livestock breeding industry. Meanwhile, the liposome has the advantages of simple preparation method, good treatment effect, high specificity, safety and non-toxicity.

Description

Technical Field

[0001] This invention relates to the field of veterinary medicine technology, and more specifically, to a folic acid-chicoric acid liposome capable of treating ulcerative colitis, its preparation and application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease commonly found in livestock farms and pet clinics, and is one of the leading causes of diarrhea in livestock and pets. In livestock farms, UC frequently occurs in weaned piglets and calves, with symptoms including diarrhea, loss of appetite, weight loss, and bloody stools. Pathological changes occur in the colon, leading to intestinal flora imbalance and consequently, decreased meat quality, resulting in significant economic losses for farms. This disease is also common in pet clinics, causing acute and chronic diarrhea in dogs and cats. Affected animals exhibit symptoms such as fever, weight loss, increased defecation frequency, and tenesmus. Pathological staining of colonic sections reveals infiltration of eosinophils, neutrophils, plasma cells, and lymphocytes. Because the etiology and pathogenesis of ulcerative colitis are not yet fully understood, there is currently a lack of effective treatments. Therefore, exploring effective treatments for ulcerative colitis is a current research focus. Clinically, the treatment of ulcerative colitis (UC) is mainly conservative, often using 5-aminosalicylic acid, steroids, immunosuppressants, and fecal microbiota transplantation to alleviate inflammation. However, these drugs all have varying degrees of adverse reactions, such as decreased appetite, osteoporosis, and metabolic disorders, and their specificity is not strong, easily leading to relapse and poor efficacy.

[0003] In summary, the existing technology has the problem that ulcerative colitis in livestock farms reduces meat quality, and the drugs used for conservative treatment all have varying degrees of adverse reactions, lack specificity, and are not very effective. There is an urgent need for a specific drug with no toxic side effects to treat ulcerative colitis. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of existing drugs for treating ulcerative colitis, the present invention aims to provide a folic acid-chicoric acid liposome for treating ulcerative colitis, as well as its preparation and application.

[0005] This folic acid-chicoric acid liposome is composed of egg yolk lecithin, cholesterol, chicoric acid, and folic acid (in a mass ratio of 10–14:2–5:1–2:1–2). It exhibits targeting properties, specifically targeting macrophages, inhibiting macrophage polarization towards the M1 type, and alleviating intestinal inflammation. This liposome demonstrates efficacy in treating ulcerative colitis in mice and also possesses advantages such as simple preparation method, excellent therapeutic effect, high specificity, and safety with no toxicity.

[0006] Folic acid and chicoric acid are both natural immunomodulators with anti-inflammatory effects. This invention uses a double-emulsion ultrasound method to prepare folic acid-chicoric acid liposomes. After characterization, the therapeutic effect on DSS-induced ulcerative colitis in mice was studied, exploring the synergistic anti-inflammatory effect of folic acid and chicoric acid, and providing a theoretical basis for the development of therapeutic drugs for ulcerative colitis.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides the application of folic acid-chicoric acid liposomes in the preparation of a medicament for treating ulcerative colitis.

[0009] This invention establishes a mouse model of ulcerative colitis by inducing inflammation in mice using sodium dextran sulfate (DSS). Results showed that folic acid-chicoric acid liposomes improved clinical symptoms (weight loss, bloody stools, etc.) in mice with ulcerative colitis, increased colon length, reduced DAI score, CMDI score, and colon index, and alleviated colonic pathological damage. After gavage administration of the liposomes, the levels of IL-1β and TNF-α in mouse serum were downregulated, while the level of the anti-inflammatory factor IL-10 in mouse serum was upregulated; the levels of TNF-α, IL-6, iNOS, and CD86 in the colon were downregulated, while the levels of the anti-inflammatory factors Arg-1 and CD206 in the mouse colon were upregulated; the polarization of macrophages towards the M1 type in the mouse colon was reduced, and the expression levels of TLR4 and NF-κB p65 proteins in the mouse colon were downregulated.

[0010] All of the above indicate that the folic acid-chicoric acid liposome has a good therapeutic effect on ulcerative colitis caused by DSS, and can provide a highly effective drug for the treatment of ulcerative colitis in animal husbandry.

[0011] More preferably, the folic acid-chicoric acid liposome is in powder form, which is dissolved in physiological saline during the experiment to prepare a liposome solution for gavage.

[0012] Preferably, the folic acid-chicoric acid liposome is composed of egg yolk lecithin, cholesterol, chicoric acid, and folic acid, and is prepared by a double emulsion ultrasonic method.

[0013] The mass ratio of egg yolk lecithin, cholesterol, chicoric acid, and folic acid is 10-14:2-5:1-2:1-2, preferably 12:3:1:1.

[0014] As a preferred embodiment, the method for preparing the folic acid-chicoric acid liposomes includes the following steps:

[0015] S1. Dissolve the folic acid modification molecule in PBS and mix thoroughly to obtain the aqueous phase; weigh egg yolk lecithin, cholesterol, and chicoric acid, dissolve them in anhydrous ethanol, and mix thoroughly to obtain the oil phase; wherein, the mass ratio of egg yolk lecithin: cholesterol: chicoric acid: folic acid is 10-14: 2-5: 1-2: 1-2 (preferably 12: 3: 1: 1);

[0016] S2. Add a small amount of aqueous phase to the oil phase for the first emulsification, and remove most of the organic solvent by rotary evaporation under reduced pressure.

[0017] S3. When a gel-like emulsion is formed, add the remaining aqueous phase for a second emulsification, and continue to remove the remaining organic solvent by rotary evaporation, so that the oil phase and the aqueous phase are fully mixed.

[0018] S4. After cooling, sonicate, filter, and freeze-dry to obtain folic acid-chicoric acid liposome powder, and store at 2-8℃ (preferably 4℃) for later use.

[0019] Preferably, in step S2, the conditions for the first emulsification are emulsification at 40℃~50℃ for 3~7 min; further, emulsification at 45℃ for 5 min;

[0020] Preferably, in step S3, the conditions for the second emulsification are emulsification at 40℃~50℃ for 8~12 min; further, emulsification at 45℃ for 10 min.

[0021] Preferably, in step S4, the ultrasound duration is 5-7 minutes; more preferably, it is 6 minutes.

[0022] The filtration process uses a 0.22μm filter membrane.

[0023] Preferably, the ulcerative colitis is ulcerative colitis induced by DSS as a pathogenic factor.

[0024] Preferably, the treatment for ulcerative colitis involves increasing body weight and decreasing the DAI score.

[0025] Preferably, the treatment of ulcerative colitis involves increasing colon length and decreasing CMDI score, colon index, and unit colon mass index.

[0026] Preferably, the treatment of ulcerative colitis aims to improve the degree of pathological damage to the colonic tissue.

[0027] Preferably, the treatment of ulcerative colitis involves inhibiting the polarization of macrophages in the colon towards the M1 type.

[0028] Preferably, the treatment of ulcerative colitis involves downregulating the levels of inflammatory factors TNF-α and IL-1β in serum and upregulating the level of anti-inflammatory factor IL-10 in serum.

[0029] Preferably, the treatment of ulcerative colitis involves downregulating the levels of inflammatory factors TNF-α, IL-6, iNOS, and CD86 in the colon, and upregulating the levels of anti-inflammatory factors Arg-1 and CD206 in the colon.

[0030] Preferably, the treatment of ulcerative colitis involves downregulating the levels of the inflammatory proteins TLR4 and NF-κBp65 in the colon.

[0031] Preferably, the drug is administered by gavage.

[0032] Preferably, the dosage of the drug for treating ulcerative colitis is 5–15 mg / kg per individual (preferably mice); the optimal dosage is 10 ± 1 mg / kg per individual (preferably mice).

[0033] A folic acid-chicoric acid liposome was prepared by the above-described preparation method.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] This invention has shown that the folic acid-chicoric acid liposome exhibits good therapeutic effects in mice with ulcerative colitis, providing a new application of folic acid-chicoric acid liposomes in the treatment of ulcerative colitis. It also provides a novel nanoliposome drug for the treatment of ulcerative colitis, laying a foundation for the development of new drugs for the treatment of ulcerative colitis. Attached Figure Description

[0036] Figure 1 The particle size (A) and electron microscopy results of folic acid-chicoric acid liposomes (B: 15000×, C: 65000×).

[0037] Figure 2 This refers to the changes in body weight of mice in each treatment group in Example 2.

[0038] Figure 3 This refers to the changes in DAI scores of mice in each treatment group in Example 2.

[0039] Figure 4 The changes were in colon length (A, B) and CMDI score (C) in mice; the model control group was compared with the blank control group: # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group: * P < 0.05; ** P < 0.01; *** P < 0.001.

[0040] Figure 5This refers to the changes in the expression of inflammatory factors in the serum of mice in each treatment group in Example 2; among them, the model control group compared with the blank control group: # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group: * P < 0.05; ** P < 0.01; *** P < 0.001.

[0041] Figure 6 This refers to the changes in body weight of mice in each treatment group in Example 3.

[0042] Figure 7 This refers to the changes in DAI scores of mice in each treatment group in Example 3.

[0043] Figure 8 The changes in colon length (A, B) and CMDI score (C) of mice in each treatment group in Example 5 are shown.

[0044] Figure 9 This refers to the changes in the polarization of colon macrophages in mice in each treatment group in Example 6.

[0045] Figure 10 These are histopathological sections of colon tissue from mice in each treatment group in Example 7; Note: (HE staining, 400×).

[0046] Figure 11 This refers to the changes in the expression of inflammatory factors in the serum of mice in each treatment group in Example 8; among them, the model control group compared with the blank control group: # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group: * P < 0.05; ** P < 0.01; *** P < 0.001.

[0047] Figure 12 This refers to the changes in mRNA expression levels of colitis-related factors in mice of each treatment group in Example 9; among them, the model control group compared with the blank control group: # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group: * P < 0.05; ** P < 0.01; *** P < 0.001.

[0048] Figure 13This refers to the changes in the expression of relevant proteins in the colon tissue of mice in each treatment group in Example 10; where NF-κB represents NF-κB p65; compared with the blank control group: # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group: * P < 0.05; ** P < 0.01; *** P < 0.001. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] 1. Preparation of folic acid-chicoric acid liposomes

[0051] S1. Dissolve the folic acid-modified molecules in PBS and mix thoroughly to obtain the aqueous phase. Weigh out egg yolk lecithin, cholesterol, and chicoric acid, dissolve them in anhydrous ethanol, and mix thoroughly to obtain the oil phase. The mass ratio of egg yolk lecithin:cholesterol:chicoric acid:folic acid is 12:3:1:1.

[0052] S2. Transfer the oil phase to a round flask, add a small amount of aqueous phase for the first emulsification (45℃, 5min), and remove most of the organic solvent (anhydrous ethanol) by rotary evaporation under reduced pressure.

[0053] S3. When a gel-like emulsion is formed, add the remaining aqueous phase for a second emulsification (45℃, 10min), and continue rotary evaporation to remove the remaining organic solvent (anhydrous ethanol) to ensure thorough mixing of the oil and aqueous phases.

[0054] S4. After cooling, the mixture was sonicated for 6 minutes and filtered through a 0.22 μm filter membrane to obtain a folic acid-chicoric acid liposome suspension. After lyophilization, the resulting powder was stored at 4℃ for later use. The encapsulation efficiency was measured to be 77.32±3.19%, and the drug loading was 16.02±0.35%. Approximately 0.16 mg of chicoric acid was contained in 1 mg of liposome powder.

[0055] Referring to the above preparation steps, (1) the difference is that chicoric acid is not added, and folic acid blank liposomes are prepared; (2) the difference is that folic acid is not added, and chicoric acid liposomes are prepared; (3) the difference is that folic acid and chicoric acid are not added, and blank liposomes are prepared.

[0056] 2. Establishment of animal models

[0057] Male C57BL / 6 mice aged 6 - 8 weeks with a body weight of 18 - 22 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the license number: SYXK(Guangdong)2022 - 0136. All operations in this experiment complied with national animal ethics regulations and were carried out at the Experimental Animal Center of South China Agricultural University, with the experimental number: 00323883. The method for establishing the model was to feed the mice with 2.5% DSS in drinking water for 7 days to establish a mouse ulcerative colitis (UC) model.

[0058] 3. Animal grouping

[0059] Mice in the blank control group (C) were given basal diet and equal amounts of pure water throughout the 1st - 14th days, and equal amounts of normal saline by gavage during the 7th - 14th days.

[0060] Mice in the model control group (M) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and equal amounts of normal saline by gavage during the 7th - 14th days.

[0061] Mice in the sulfasalazine group (S) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and gavaged with 100 mg / kg of sulfasalazine during the 7th - 14th days.

[0062] Mice in the blank liposome group (K) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and gavaged with 10 mg / kg of blank liposome during the 7th - 14th days.

[0063] Mice in the chicoric acid group (CA) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and gavaged with 10 mg / kg of chicoric acid during the 7th - 14th days.

[0064] Mice in the folic acid blank liposome group (FK) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and gavaged with 10 mg / kg of folic acid blank liposome during the 7th - 14th days.

[0065] Mice in the chicoric acid liposome group (CL) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and gavaged with 10 mg / kg of chicoric acid liposome during the 7th - 14th days.

[0066] Mice in the folic acid - chicoric acid liposome group (FC) were given basal diet, fed with 2.5% DSS in drinking water during the 1st - 7th days, and gavaged with 10 mg / kg of folic acid - chicoric acid liposome during the 7th - 14th days.

[0067] For each liposome group, normal saline was added for dissolution during the experiment to prepare a liposome solution for gavage.

[0068] Example 1 Characterization of folic acid - chicoric acid liposome

[0069] 1. Experimental Methods

[0070] Folic acid-chicoric acid liposome powder was evenly spread on conductive adhesive and mounted on a conductive stage. After gold plating, its morphology was observed under a scanning electron microscope at 15,000x and 65,000x magnification. The folic acid-chicoric acid liposome powder was then dissolved in pure water, and the particle size, zeta potential, and polydispersity index of the folic acid-chicoric acid liposomes were determined using a nano-laser particle size analyzer.

[0071] 2. Experimental Results

[0072] like Figure 1 As shown, the folic acid-chicoric acid liposomes mainly have a particle size between 100 and 200 nm, with a relatively concentrated particle size distribution. The average particle size is 120.40 ± 0.46 nm, the average potential is -1.72 mV, and the polydispersity index is 0.24. Under electron microscopy, most of them are spherical with smooth surfaces, small particle size, and good uniformity in particle size.

[0073] Example 2: Screening of folic acid-chicoric acid liposome dosage

[0074] 1. Experimental Methods

[0075] Based on the human-to-mouse body surface area equivalent dose ratio of 0.0025, the low-dose, medium-dose, and high-dose groups of folic acid-chicoric acid liposomes were determined to be 5, 10, and 15 mg / kg, respectively. The experiment consisted of 5 groups, with 5 mice in each group: blank control group (C), model control group (M), low-dose folic acid-chicoric acid liposome group (FL), medium-dose folic acid-chicoric acid liposome group (FM), and high-dose folic acid-chicoric acid liposome group (FH). Six- to eight-week-old male C57BL / 6 mice weighing 18–20 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were fed 2.5% DSS in drinking water for 7 days, followed by gavage administration of the drugs once daily for 7 days. During this period, mouse weight, DAI score, colonic index and unit mass index, colonic length, CMDI score, and serum inflammatory factor expression were recorded.

[0076] 2. Experimental Results

[0077] Changes in mouse body weight during the experiment, such as Figure 2 As shown, on day 6 after modeling, compared with the blank control group, the body weight of mice in each group decreased significantly (P<0.01), and the body weight of mice in the model control group was the lowest during the drug administration period; compared with the model control group, the body weight of mice in each drug administration group showed an increasing trend, with the FM group having the highest body weight at day 14.

[0078] Mouse DAI score during the experiment: Figure 3As shown, from day 1 of modeling, compared with the blank control group, the DAI scores of mice in each group were significantly increased (P < 0.01); compared with the model control group, the DAI scores of mice in each drug-treated group were significantly decreased (P < 0.05), reaching the lowest point on day 7 of drug administration.

[0079] As shown in Table 1, compared with the blank control group, the colon index and colon mass index of mice in the model control group were significantly increased (P<0.05); compared with the model control group, the colon index and colon mass index of mice in the FM group were significantly decreased (P<0.05).

[0080] like Figure 4 As shown, compared with the blank control group, the colon length of mice in the model control group was significantly decreased (P<0.01) and the CMDI score was significantly increased (P<0.001); compared with the model control group, the colon length of mice in the FM group was significantly increased (P<0.05) and the CMDI score was the lowest (P<0.01).

[0081] like Figure 5 As shown, compared with the blank control group, the serum IL-1β level in the model control group mice was significantly increased (P<0.05), and the IL-10 level was significantly decreased (P<0.001); compared with the model control group, the serum IL-1β level in the FM group mice was significantly decreased (P<0.001), and the IL-10 level was significantly increased (P<0.01).

[0082] The results showed that low (5 mg / kg), medium (10 mg / kg), and high (15 mg / kg) doses of folic acid-chicoric acid liposomes all slowed weight loss in ulcerative colitis (UC) mice, reduced DAI and CMDI scores, increased colon length, and decreased colonic index. Furthermore, they reduced serum IL-1β expression and increased IL-10 expression in UC mice. The FM group showed a more significant therapeutic effect on ulcerative colitis mice than the FL and FH groups; therefore, the medium dose (10 mg / kg) of folic acid-chicoric acid liposomes was selected for subsequent experiments.

[0083] Table 1. Mouse colonic index and colonic mass index per unit (g, X±SD, n=5)

[0084]

[0085] Note: If the superscript letters of the values ​​in the table are the same, it indicates that there is no significant difference between the groups (P > 0.05). If the letters are different, it indicates that there is a significant difference between the groups (P < 0.05). "a" represents the maximum value, and "b", "c", and "d" represent values ​​in descending order; the same applies below.

[0086] Example 3: Mouse body weight and DAI score in each group

[0087] 1. Experimental Methods

[0088] Following step 1 of Example 2, 6-8 week old male C57BL / 6 mice weighing 20-22g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experiment was divided into 8 groups, with 6 mice in each group: blank control group (C), model control group (M), sulfasalazine group (S), blank liposome group (K), chicoric acid group (CA), folic acid blank liposome group (FK), chicoric acid liposome group (CL), and folic acid-chicoric acid liposome group (FC). Mice were weighed, and daily weight changes, fecal viscosity, and fecal occult blood were recorded. The DAI score for each group was calculated, as detailed in Table 2. The DAI score was calculated as follows: DAI score = (percentage weight loss score + fecal viscosity score + fecal occult blood score) / 3.

[0089] Table 2. Detailed criteria for DAI scoring in each group of mice.

[0090]

[0091] 2. Experimental Results

[0092] Changes in body weight of mice in each group during the experiment are as follows: Figure 6 As shown, compared with the blank control group, the body weight of mice in each model group decreased significantly (P<0.05), reaching its lowest point between day 7 and day 11, and then gradually increased. At the end of treatment, the body weight of mice in the S group and FC group was significantly higher than that of the model control group (P<0.05), and there was no significant difference from the blank control group.

[0093] Mouse DAI score during the experiment: Figure 7 As shown, compared with the blank control group, the DAI scores of mice in each group were significantly increased after modeling (P<0.05), and reached the maximum value between day 6 and day 7; after drug administration, the DAI scores of mice in each group gradually decreased, reaching the lowest value on day 7 of drug administration.

[0094] Example 4: Colonic index and colonic mass index of mice in each group

[0095] 1. Experimental Methods

[0096] The experimental groups followed the procedure in Example 3, step 1. After the drug treatment was completed, animal specimens were collected after a 24-hour fast. Mice were weighed, anesthetized, and euthanized by cervical dislocation. The colons were then weighed. Based on the mouse body weight, colon weight, colon length, and colon weight, the colonic index and colon mass index per unit area were calculated for each group of mice.

[0097] Colon index = Colon weight (g) / Body weight (g) × 100%

[0098] Unit colon mass index = colon mass (g) / colon length (cm) × 100%

[0099] 2. Experimental Results

[0100] The results are shown in Table 3. Compared with the blank control group, the colon index and colon mass index of mice in the model control group were significantly increased (P<0.05); compared with the model control group, the colon index and colon mass index of mice in the FC group were significantly decreased (P<0.05).

[0101] Table 3. Mouse colonic index and colonic mass index per unit (g, X±SD, n=6)

[0102]

[0103] Example 5: Changes in colon length and CMDI score in mice of different groups

[0104] 1. Experimental Methods

[0105] The experimental groups were determined according to step 1 of Example 3. After sampling, mouse colons were taken, colon length was measured, a portion of the colon was cut off, and the degree of colon tissue damage was observed visually. The Macroscopic Colon Damage Index (CMDI) was calculated according to the scoring table, as shown in Table 4.

[0106] Table 4. Detailed CCMDI scoring criteria for each group of mice.

[0107]

[0108] 2. Experimental Results

[0109] The results are as follows Figure 8 As shown, compared with the blank control group, the colon length of mice in the model control group was significantly decreased (P<0.001) and the CMDI score was significantly increased (P<0.001); compared with the model control group, the colon length of mice in the FC group was significantly increased (P<0.001) and the CMDI score was significantly decreased (P<0.001).

[0110] Example 6: Detection of macrophage polarization in the colon of mice in each group

[0111] 1. Experimental Methods

[0112] The experimental grouping followed step 1 of Example 3. Colonic segments were thoroughly ground in pre-cooled PBS and filtered through a 200-mesh filter to obtain a colonic single-cell suspension. The cell density was adjusted to 1 × 10⁻⁶ cells / cells. 6Cells were collected at a density of [number] cells / mL and transferred to centrifuge tubes. F4 / 80 antibody, CD11b antibody, and CD86 antibody were added, and the cells were incubated at 4°C for 30 min for surface staining. After incubation, the cells were washed with PBS, fixed with cell fixation solution at room temperature in the dark for 30 min, centrifuged at 150g for 5 min, resuspended in permeabilization buffer, and CD206 antibody was added. After mixing, the cells were incubated at room temperature in the dark for 30 min. After incubation, the cells were washed twice, and finally resuspended in 300 μL of PBS. The cells were then transferred to flow cytometry tubes for analysis using flow cytometry. F4 / 80 is a cell marker for macrophages, CD11b is a cell marker for neutrophils, CD86 is a cell marker for M1 macrophages, and CD206 is a cell marker for M2 macrophages.

[0113] 2. Experimental Results

[0114] Macrophages from the colon tissue of mice in each group were screened by flow cytometry for subsequent staining analysis. The macrophage polarization results are as follows: Figure 9 As shown, compared with the blank control group, the levels of the M1 marker CD86 and the M2 marker CD206 in the macrophages of the model control group mice were significantly increased (P < 0.001), and the ratio of M1 / M2 macrophages in the model control group mice was significantly increased (P < 0.001). Compared with the model control group, the levels of CD86 in the FC group mice were significantly decreased (P < 0.001), the levels of CD206 were significantly increased (P < 0.001), and the M1 / M2 ratio was significantly decreased (P < 0.001).

[0115] Example 7: Observation of histopathological sections of colon tissue from each group of mice

[0116] 1. Experimental Methods

[0117] The experimental groups were determined according to step 1 of Example 3. After sampling, colon tissue from each group of mice was cut, fixed in 10% neutral formaldehyde solution for 24 hours, then eluted with ethanol in a gradient, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and the pathological damage of the colon tissue of each group of mice was observed under an optical microscope.

[0118] 2. Experimental Results

[0119] The microstructure of the colon tissue of each group of mice was observed under an optical microscope. For example... Figure 10 As shown, in group C, the colonic villi were neatly arranged and structurally intact, with no obvious pathological changes or inflammatory cell proliferation and infiltration; in group M, the colonic mucosa was irregularly arranged, structurally damaged, and showed extensive inflammatory cell infiltration; in group FC, the colonic villi were neatly arranged and structurally intact, with no obvious pathological changes or inflammatory cell infiltration. The section results showed that the colonic mucosal damage and inflammation in group FC were significantly improved.

[0120] Example 8: Determination of serum inflammatory factor expression in mice of different groups

[0121] 1. Experimental Methods

[0122] The experimental groups followed the steps in Example 3, Step 1. After anesthetizing the mice, the eyeballs were removed and blood was collected in non-anticoagulated blood collection tubes. The plasma samples were placed in a centrifuge and centrifuged at 3500 r / min for 8 min. The supernatant was collected, and the levels of IL-1β, IL-10, and TNF-α in the mouse serum were measured using ELISA.

[0123] 2. Experimental Results

[0124] The results are as follows Figure 11 As shown, compared with the blank control group, the serum TNF-α content of mice in the model control group was significantly increased (P<0.001), and the IL-10 content showed a decreasing trend; compared with the model control group, the serum TNF-α and IL-1β content of mice in the FC group was significantly decreased (P<0.001, P<0.01), and the IL-10 content showed an increasing trend.

[0125] Example 9: Detection of mRNA expression levels of inflammatory factors in the colon of mice in each group

[0126] 1. Experimental Methods

[0127] The experimental grouping followed step 1 of Example 3. After homogenizing the colon tissue, centrifuging was performed to collect the supernatant. Total RNA was extracted, and cDNA was synthesized via reverse transcription. Finally, quantitative RT-PCR was performed using pre-designed primers (primer sequences are shown in Table 5). 2 -ΔΔCT The relative quantitative analysis was performed to determine the mRNA expression levels of TNF-α, IL-6, iNOS, CD86, Arg-1, and CD206 in the colon.

[0128] Table 5 Primer sequence list

[0129]

[0130] 2. Experimental Results

[0131] The results are as follows Figure 12As shown, compared with the blank control group, the mRNA expression levels of TNF-α, IL-6, iNOS, and CD86 in the colon of mice in the model control group were significantly increased (P < 0.01); the mRNA expression levels of Arg-1 and CD206 showed a decreasing trend. Compared with the model control group, the mRNA expression levels of CD86, TNF-α, and iNOS in mice in the FC group were significantly decreased (P < 0.01, P < 0.05, P < 0.05), and the mRNA expression level of IL-6 showed a decreasing trend; the mRNA expression level of Arg-1 was significantly increased (P < 0.001), and the mRNA expression level of CD206 showed an increasing trend.

[0132] Example 10: Detection of inflammatory-related protein expression in the colon of mice in each group

[0133] 1. Experimental Methods

[0134] The experimental groups followed the steps in Example 3, section 1. Total protein was extracted from colon tissue, and the protein concentration was determined using the BCA method. An 8% SDS-PAGE gel was prepared, and after loading the sample, electrophoresis, transfer to a membrane, blocking, incubation with primary antibody and secondary antibody were performed. After development with chemiluminescence solution, the grayscale values ​​of the sample protein bands were analyzed using ImageJ software to detect the expression levels of TLR4 and NF-κB p65 proteins in cells.

[0135] 2. Experimental Results

[0136] The results are as follows Figure 13 As shown, compared with the blank control group, the expression levels of TLR4 and NF-κBp65 proteins in the colon of mice in the model control group were significantly increased (P < 0.001, P < 0.01); compared with the model control group, the expression levels of TLR4 and NF-κBp65 proteins in the colon of mice in the FC group were significantly decreased (P < 0.001).

[0137] In summary, mice with ulcerative colitis treated with folic acid-chicoric acid liposomes showed superior performance compared to other single-drug groups in terms of weight change, colon length, DAI score, CMDI score, colon index, macrophage polarization, colonic tissue pathological damage, and anti-inflammatory effects. This indicates that folic acid-chicoric acid liposomes have a good therapeutic effect on DSS-induced ulcerative colitis, and that folic acid and chicoric acid have a certain synergistic effect in alleviating inflammation in mice with ulcerative colitis. The main components of folic acid-chicoric acid liposomes are all derived from natural ingredients, making them safe and non-toxic. They can improve the quality of livestock and poultry meat and reduce food safety risks. Therefore, folic acid-chicoric acid liposomes can provide a new approach for the treatment of ulcerative colitis in livestock farming.

[0138] Furthermore, through numerous experiments, the inventors discovered that folic acid-chicoric acid liposomes prepared by weighing the raw materials in a mass ratio of egg yolk lecithin:cholesterol:chicoric acid:folic acid of 10–14:2–5:1–2:1–2 resulted in higher encapsulation rates, with a mass ratio of 12:3:1:1 being the optimal one. Dosage screening revealed that folic acid-chicoric acid liposomes at doses of 5–15 mg / kg were effective, with a gavage administration of 10 mg / kg maximizing the drug's cost-effectiveness.

[0139] The above embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

[0140] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of folate-chicoric acid liposomes for the preparation of a medicament for the treatment of ulcerative colitis, characterized in that: The folate-chicoric acid liposome is prepared by a multiple emulsion ultrasonic method and is composed of egg yolk lecithin, cholesterol, chicoric acid and folate; the mass ratio of the egg yolk lecithin, cholesterol, chicoric acid and folate is 10-14:2-5:1-2:1-2.

2. The use according to claim 1, wherein: The mass ratio of the egg yolk lecithin, cholesterol, chicoric acid and folate is 12:3:1:

1.

3. The use according to claim 1 or 2, wherein: The ulcerative colitis is induced by DSS as a pathogenic factor; The drug is administered by gavage.

4. The use according to claim 1 or 2, wherein: The drug for treating ulcerative colitis is administered at a dose of 5-15 mg / kg of the individual.

5. Use according to claim 1 or 2, characterized in that: for at least one of the following: a) the treatment of ulcerative colitis is to increase body weight; to decrease DAI score; b) the treatment of ulcerative colitis is to increase colon length; to decrease CMDI score, colon index, unit colon mass index; c) the treatment of ulcerative colitis is to improve the degree of pathological damage of colon tissue; d) the treatment of ulcerative colitis is to inhibit the polarization of macrophages in the colon to M1 type; e) the treatment of ulcerative colitis is to decrease the level of inflammatory factors TNF-α, IL-1β in serum; to increase the level of anti-inflammatory factor IL-10 in serum; f) the treatment of ulcerative colitis is to decrease the level of inflammatory factors TNF-α, IL-6, iNOS, CD86 in the colon; to increase the level of anti-inflammatory factors Arg-1, CD206 in the colon; g) the treatment of ulcerative colitis is to decrease the level of inflammatory proteins TLR4 protein, NF-κB p65 protein in the colon.

6. A process for the preparation of folate-oxylate liposomes as claimed in any one of claims 1 to 5, characterized in that: comprising the following steps: S1. Dissolve the folate-modified molecule in PBS and mix well to obtain an aqueous phase; weigh the egg yolk lecithin, cholesterol and chicoric acid, dissolve them in anhydrous ethanol and mix well to obtain an oil phase; S2. Add a small amount of the aqueous phase to the oil phase for the first emulsification, and remove most of the organic solvent by rotary evaporation under reduced pressure; S3. When a colloidal emulsion is formed, add the remaining aqueous phase for the second emulsification, and continue to remove the remaining organic solvent by rotary evaporation to fully mix the oil phase and the aqueous phase; S4. After cooling, ultrasonic, filtration, and freeze-drying to obtain folate-chicoric acid liposome powder.

7. The preparation method according to claim 6, wherein: In step S2, the first emulsification is performed at 40-50℃ for 3-7 min; In step S3, the second emulsification is performed at 40-50℃ for 8-12 min; In step S4, the ultrasonic time is 5-7 min; The filtration is through a 0.22μm filter membrane.

8. A folate-erucic acid liposome, characterized by: prepared by the preparation method of any one of claims 6-7.

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