Folic acid-echinacea source alkyl amide liposome for treating ulcerative colitis as well as preparation and application of folic acid-echinacea source alkyl amide liposome

Abstract

The invention discloses a folic acid-echinacea source alkyl amide liposome for treating ulcerative colitis as well as preparation and application of the folic acid-echinacea source alkyl amide liposome. The folic acid-echinacea source alkylamide liposome is prepared from egg yolk lecithin, cholesterol, echinacea source alkylamide and phospholipid-polyethylene glycol-folic acid (the mass ratio is (4-8): (2-5): (1-2): (1-2)), has targeting property and macrophage targeting effect, and can improve clinical symptoms (weight loss, bloody stool and the like) of mice with ulcerative colitis, increase the colon length and improve the ulcerative colitis activity. The DAI score and the indexes of all organs and colon are reduced, the colon pathological injury is relieved, and the intestinal inflammation is relieved. The liposome shows a good effect of treating mice with ulcerative colitis, a novel nano-liposome drug is provided for treatment of ulcerative colitis, and a certain foundation is laid for research and development of new drugs for treating ulcerative colitis. Meanwhile, the preparation method has the advantages of being simple, good in treatment effect, high in specificity, safe and non-toxic.

Description

Technical Field

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

[0002] Ulcerative colitis (UC) is a chronic, nonspecific, and inflammatory disease characterized by inflammation and ulceration of the colorectal mucosa. Patients often present with clinical symptoms such as weight loss, decreased appetite, diarrhea, abdominal pain, and bloody or mucoid stools. Pathological changes in the colonic tissue include inflammation, bleeding, mucosal ulceration, and mucosal erosion. Influenced by environmental and dietary factors (such as high-fat, high-sugar diets), immune factors, and intestinal flora imbalance, the incidence of UC has been increasing year by year in recent years. UC is also common in livestock and poultry farms and is one of the causes of diarrhea in livestock and poultry. This disease often occurs in piglets and calves, causing symptoms such as chronic diarrhea, weight loss, decreased appetite, and rough coat, leading to reduced productivity and significant economic losses in livestock and poultry farming. In canines and cats, affected pets may experience acute or chronic diarrhea, tenesmus, bloody stools, weight loss, and depression, and may even develop colon tumors. The etiology and pathogenesis of ulcerative colitis (UC) are not yet fully understood. However, years of research have generally led to the conclusion that UC development is related to immune dysregulation, intestinal barrier damage, gut microbiota imbalance, and environmental factors, and is a complex process involving multiple factors. Besides surgery, UC treatment primarily involves conservative drug therapy, typically using 5-aminosalicylic acid, glucocorticoids, and immunosuppressants to improve UC through anti-inflammatory and immunomodulatory effects. However, these drugs all have varying degrees of adverse reactions, including allergic reactions, kidney damage, and even exacerbation of symptoms such as abdominal pain and diarrhea, resulting in poor treatment outcomes. In recent years, research into non-pharmacological treatments for UC, such as femoral head thrombectomy (FMT) and stem cell therapy, has been flourishing and can, to some extent, compensate for the shortcomings of drug therapy.

[0003] In summary, while traditional treatments can improve symptoms, their toxic side effects should not be underestimated, as they can easily worsen bodily damage and reduce meat quality in livestock. Therefore, researching novel, effective, and safe drugs for treating ulcerative colitis (UC) has promising applications. 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-echinacea-derived alkylamide liposome for treating ulcerative colitis, as well as its preparation and application.

[0005] This folic acid-echinacea-derived alkylamide liposome is composed of egg yolk lecithin, cholesterol, alkylamide, phospholipid-polyethylene glycol-folic acid (mass ratio 4–8:2–5:1–2:1–2). It exhibits targeting properties, specifically targeting macrophages, inhibiting macrophage polarization, and alleviating intestinal inflammation. This folic acid-modified liposome demonstrates therapeutic efficacy in mice with ulcerative colitis, and also possesses advantages such as simple preparation method, excellent therapeutic effect, high specificity, and safety with no toxicity.

[0006] Folic acid and echinacea-derived alkylamides are both natural immunomodulators with anti-inflammatory effects. This invention uses an ethanol injection method to prepare folic acid-echinacea-derived alkylamide 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 echinacea-derived alkylamides, 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-echinacea-derived alkylamide 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 dextran sulfate sodium salt (DSS). Results showed that folic acid-echinacea-derived alkylamide liposomes improved clinical symptoms (weight loss, bloody stools, etc.) in mice with ulcerative colitis, increased colon length, reduced DAI scores, and decreased organ and colonic indices, alleviating colonic pathological damage. After gavage administration of these liposomes, the levels of TNF-α, IL-1β, IL-6, and COX2 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, IL-1β, iNOS, COX2, and CD86 in the colon were downregulated, while the levels of the anti-inflammatory factors Arg-1, IL-10, and CD206 in the mouse colon were upregulated; and the colonic tissue-related signaling pathways NF-κB, JAK2, and STAT3 were significantly downregulated, while Nrf2 and HO-1 were significantly upregulated.

[0010] All of the above indicate that the folic acid-echinacea-derived alkylamide 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-echinacea-derived alkylamide liposomes are in powder form, which are dissolved in 0.5% CMC-Na solution during the experiment to prepare a liposome solution for gavage.

[0012] Preferably, the folic acid-echinacea-derived alkylamide liposome is composed of egg yolk lecithin, cholesterol, echinacea-derived alkylamide, and phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA), and is prepared by ethanol injection method.

[0013] The mass ratio of egg yolk lecithin, cholesterol, echinacea-derived alkylamide, phospholipid-polyethylene glycol-folic acid is 4-8:2-5:1-2:1-2, preferably 6:3:1:1.

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

[0015] S1. Echinacea root powder was soaked and extracted with methanol, the methanol extract was collected, and the extract was concentrated under reduced pressure to obtain crude extract of Echinacea root methanol extract.

[0016] S2. Mix the crude extract with pure water by vortexing, extract three times with an equal volume of petroleum ether, collect the upper extract, concentrate under reduced pressure to obtain the petroleum ether extract, freeze-dry and weigh for later use.

[0017] S3. Silica gel column chromatography, collect the eluent for thin-layer chromatography analysis, and use an ultraviolet analyzer to detect the eluent that has parallel colored spots with the standard at a wavelength of 254 nm. Then, concentrate under reduced pressure and evaporate the solvent to obtain the purified alkylamide, namely echinacea-derived alkylamide.

[0018] S4. Dissolve phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA) in a small amount of dichloromethane, evaporate to dryness to form a film, dissolve in PBS and mix thoroughly to obtain the aqueous phase; weigh egg yolk lecithin, cholesterol, and echinacea-derived alkylamide and dissolve in anhydrous ethanol, mix thoroughly to obtain the oil phase; wherein, the mass ratio of egg yolk lecithin: cholesterol: echinacea-derived alkylamide: phospholipid-polyethylene glycol-folic acid is 4-8: 2-5: 1-2: 1-2 (preferably 6: 3: 1: 1);

[0019] S5. Slowly inject the oil phase into the aqueous phase and continue stirring to evaporate the ethanol and remove most of the organic solvent;

[0020] S6. Continue stirring to evaporate and remove the remaining organic solvent, so that the oil phase and water phase are fully mixed;

[0021] S7. After cooling, sonicate, filter, and freeze-dry to obtain folic acid-echinacea-derived alkylamide liposome powder, which is stored at -20℃ for later use.

[0022] Preferably, in step S1, the echinacea root powder is soaked and mixed with methanol, sonicated for 15-25 min (preferably 20 min), and extracted three times at room temperature (7-9, 3-5, 3-5 days) (preferably 8 days, 4 days, 4 days). The methanol extracts are collected and combined by filtration, and concentrated under reduced pressure at 45-55℃ (preferably 50℃) to obtain a crude extract of echinacea root methanol extract.

[0023] Preferably, in step S2, the crude extract is vortexed with pure water, extracted three times with an equal volume of petroleum ether (boiling point 60-90°C), the upper extract is collected, and concentrated under reduced pressure at 45-55°C (preferably 50°C) to obtain the petroleum ether extract.

[0024] Preferably, in step S3, the eluent used for elution is a gradient mixed solvent of n-hexane and ethyl acetate; further, elution is performed using a n-hexane:ethyl acetate ratio (9:1→5:1→3:1, v / v).

[0025] Preferably, in step S4, the preparation method of the phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA) includes the following steps: reacting activated folic acid with NH2-PEG-DSPE at room temperature in the dark, dialyzing, filtering, and drying to obtain phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA).

[0026] Furthermore, the activated folic acid is obtained by activating folic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS); preferably, the molar ratio of folic acid:EDC:NHS is 1:2:2; and the activation time is 4-5 hours.

[0027] Furthermore, the molecular weight of the NH2-PEG-DSPE is 2000; the ratio of folic acid to NH2-PEG-DSPE is 0.1 nmol: 50 mg; and the reaction time at room temperature in the dark is 22–26 h (preferably 24 h).

[0028] Furthermore, dialysis was performed using deionized water as the dialysis medium in the dark for 48 hours, with the dialysis medium being replaced at 2, 4, 8, 12, 24, and 48 hours respectively; filtration was performed using a 0.22 μm aqueous phase filter membrane; and drying was performed by freeze drying.

[0029] Preferably, in step S5, the continuous stirring conditions are 400-600 r / min for 10-20 min; more preferably, 500 r / min for 15 min.

[0030] Preferably, in step S6, the stirring speed is 400-600 r / min; more preferably 500 r / min.

[0031] Preferably, in step S7, the ultrasound duration is 5-7 minutes; more preferably, it is 6 minutes.

[0032] The filtration process involves passing the solution through a 0.22 μm filter membrane to obtain a uniform, sterile folic acid-echinacea-derived alkylamide liposome suspension.

[0033] Preferably, the ulcerative colitis is ulcerative colitis induced by DSS as a pathogenic factor.

[0034] Preferably, the treatment for ulcerative colitis involves increasing body weight and decreasing the DAI score.

[0035] Preferably, the treatment of ulcerative colitis involves increasing colon length; decreasing heart, liver, spleen, and colon indices; and increasing lung and kidney indices.

[0036] Preferably, the treatment of ulcerative colitis aims to improve the degree of pathological damage and inflammatory response of colonic tissue.

[0037] Preferably, the treatment of ulcerative colitis involves downregulating the levels of inflammatory factors TNF-α, IL-1β, IL-6, and COX2 in the serum, and upregulating the level of the anti-inflammatory factor IL-10 in the serum.

[0038] Preferably, the treatment of ulcerative colitis involves downregulating the levels of inflammatory factors TNF-α, IL-6, IL-1β, iNOS, COX2, and CD86 in the colon, and upregulating the levels of anti-inflammatory factors IL-10, Arg-1, and CD206 in the colon.

[0039] Preferably, the treatment of ulcerative colitis involves downregulating the mRNA expression levels of related signaling pathways NF-κB, JAK2, and STAT3 in the colon, and upregulating the mRNA expression levels of related signaling pathways Nrf2 and HO-1 in the colon.

[0040] Preferably, the drug is administered by gavage.

[0041] Preferably, the dosage of the drug for treating ulcerative colitis is 2–8 mg / kg per individual (preferably mice); the optimal dosage is 8 ± 1 mg / kg per individual (preferably mice).

[0042] A folic acid-echinacea-derived alkylamide liposome was prepared by the above-described method.

[0043] The present invention has the following advantages and effects compared with the prior art:

[0044] This invention has found that preparing echinacea-derived alkylamides into echinacea-derived alkylamide liposomes or folic acid-echinacea-derived alkylamide liposomes can better exert the therapeutic effect on ulcerative colitis. Among them, folic acid-echinacea-derived alkylamide liposomes showed the best therapeutic effect. These folic acid-echinacea-derived alkylamide liposomes exhibited good therapeutic effects in mice with ulcerative colitis, providing a novel nanoliposome drug for the treatment of ulcerative colitis and laying a foundation for the development of new drugs for the treatment of ulcerative colitis. Attached Figure Description

[0045] Figure 1 This is the technical route for the extraction and purification of alkylamide compounds from Echinacea roots in Example 1.

[0046] Figure 2 These are HPLC chromatograms of the alkylamide reference standard (a), petroleum ether extract (b), and purified alkylamide (c) from Example 2.

[0047] Figure 3 This is a liquid chromatography-mass chromatogram of the purified alkylamide from Example 2.

[0048] Figure 4 These are primary mass spectra of compounds (Cpd) 1, 2 and 5 in the alkylamide purified in Example 2.

[0049] Figure 5 The results are the scanning electron microscopy results (24927×, 79965×) of the folic acid-echinacea-derived alkylamide liposomes in Example 3.

[0050] Figure 6 The results are obtained from transmission electron microscopy observations of the folic acid-echinacea-derived alkylamide liposomes in Example 3.

[0051] Figure 7 The results show the particle size distribution of the folic acid-echinacea-derived alkylamide liposomes in Example 3.

[0052] Figure 8 This is an HPLC chromatogram of the alkylamide reference standard (a) and the folic acid-echinacea-derived alkylamide liposome test sample (b) in Example 4.

[0053] Figure 9 The particle size and potential changes of the folic acid-echinacea-derived alkylamide liposomes in Example 5 at different temperatures and times are shown.

[0054] Figure 10 This refers to the change in the release rate of folic acid-echinacea-derived alkylamide liposomes under different time and pH conditions in Example 5.

[0055] Figure 11 This refers to the changes in body weight of mice in each treatment group in Example 6.

[0056] Figure 12 This refers to the changes in DAI scores of mice in each treatment group in Example 6.

[0057] Figure 13 This refers to the change in colon length in mice in Example 7; where the model control group (M) is compared with the blank control group (C): # P<0.05; ## P<0.01, ### P<0.001; Compared with the model control group (M): * P<0.05; ** P<0.01, ***P<0.001.

[0058] Figure 14 These are histopathological observations of colon tissue sections from mice in each treatment group in Example 8; Note: (HE staining, 40×, black arrows indicate lesion sites), scale bar: 100μm.

[0059] Figure 15 The changes in the expression of inflammatory factors in the serum of mice in each treatment group in Example 9; among them, the model control group (M) compared with the blank control group (C): # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group (M): * P < 0.05; ** P < 0.01; *** P < 0.001.

[0060] Figure 16 This refers to the changes in mRNA expression levels of inflammatory factors in the colon tissue of mice in each treatment group in Example 10; among them, the model control group (M) is compared with the blank control group (C): # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group (M): * P < 0.05; ** P < 0.01; *** P < 0.001.

[0061] Figure 17 This refers to the changes in mRNA expression levels of related signaling pathways in the colon tissue of mice in each treatment group in Example 11; among them, the model control group (M) compared with the blank control group (C): # P < 0.05; ## P < 0.01; ### P < 0.001; Compared with the model control group (M): * P < 0.05;** P < 0.01; *** P < 0.001. Detailed Implementation

[0062] 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.

[0063] 1. Preparation of folic acid-echinacea-derived alkylamide liposomes

[0064] S1. Weigh 2.5 kg of Echinacea root powder, soak and mix it with methanol, sonicate for 20 min, and then extract it three times at room temperature (8, 4 and 4 days respectively). Filter, collect and combine the methanol extracts, and concentrate them under reduced pressure at 50℃ to obtain crude extract of Echinacea root methanol extract.

[0065] S2. The crude extract was vortexed with pure water and extracted three times with an equal volume of petroleum ether (boiling point 60-90℃). The upper extract was collected and concentrated under reduced pressure at 50℃ to obtain the petroleum ether extract, which was then freeze-dried and weighed for later use.

[0066] S3. Purification was performed using silica gel column chromatography with hexane:ethyl acetate as the eluent for the petroleum ether extract of Echinacea root. The hexane:ethyl acetate ratio was continuously adjusted (9:1→5:1→3:1, v / v), and the eluent was collected based on the eluted bands. The collected eluent was analyzed by thin-layer chromatography (TLC) with hexane:ethyl acetate (3:1, v / v) as the developing solvent. The results were detected using a UV analyzer and compared with the standard. The eluent with parallel spots to the standard at 254 nm was collected, and then concentrated under reduced pressure and evaporated to obtain the target purified product: Echinacea-derived alkylamide.

[0067] S4. Folic acid-modified alkylamide liposomes were prepared using the ethanol injection method. An appropriate amount of phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA) was dissolved in a small amount of dichloromethane, evaporated to dryness to form a film, and then hydrated with 30 mL of PBS to obtain the aqueous phase. Egg yolk lecithin, cholesterol, and echinacea-derived alkylamide were weighed and dissolved in anhydrous ethanol, and mixed thoroughly to obtain the oil phase. The mass ratio of egg yolk lecithin:cholesterol:echinacea-derived alkylamide:phospholipid-polyethylene glycol-folic acid was 6:3:1:1.

[0068] S5. Place the aqueous phase on a magnetic stirrer (500 r / min), slowly inject the oil phase and continue stirring for 15 min, then evaporate the ethanol to remove most of the organic solvent.

[0069] S6. Place in a fume hood and stir continuously to evaporate and remove the remaining organic solvent, so that the oil phase and water phase are fully mixed.

[0070] S7. After cooling, the suspension was sonicated for 6 minutes and then filtered through a 0.22 μm hydrophilic membrane to obtain a homogeneous and sterile folic acid-echinacea-derived alkylamide liposome suspension. The suspension was lyophilized to obtain a powder and stored at -20℃ for later use. The encapsulation efficiency was measured to be 83.04% ± 1.73%, and the drug loading was 4.54% ± 0.12% (referring to the content of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide).

[0071] In this preparation, the folic acid blank liposomes (FL) differ from the above in that no echinacea-derived alkylamide is added, thus obtaining folic acid blank liposomes; the echinacea-derived alkylamide liposomes (AL) differ from the above in that no folic acid is added, thus obtaining echinacea-derived alkylamide liposomes.

[0072] The preparation method of the phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA) includes the following steps:

[0073] (1) Accurately weigh a certain amount of folic acid (0.0441g, 0.1nmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (0.0310g / 35μL, 0.2nmol) and N-hydroxysuccinimide (NHS) (0.0230g, 0.2nmol) according to a molar ratio of 1:2:2, place them in a beaker, add 10mL of DMSO, and activate at room temperature in the dark for 4h under magnetic stirring. Add 50mg of NH2-PEG-DSPE (molecular weight: 2000) and continue the reaction at room temperature in the dark for 24h.

[0074] (2) The prepared solution, after reacting for 24 hours, was transferred to a dialysis bag and dialyzed for 48 hours in the dark using deionized water as the dialysis medium (magnetic stirring is not required). The dialysis medium was changed at 2, 4, 8, 12, 24, and 48 hours. The final dialysate (which should be a pale yellow liquid) was filtered through a 0.22 μm aqueous filter membrane, freeze-dried, and a pale yellow, dried phospholipid-polyethylene glycol-folic acid (DSPE-PEG-FA) was obtained and stored for later use.

[0075] 2. Establishment of animal models

[0076] Five-week-old SPF-grade male KM mice weighing 21-33 g were purchased from Zhuhai Bestone Biotechnology Co., Ltd., with the license number: SYXK(Yue)2020-0051. The mice were housed in the Experimental Animal Center of South China Agricultural University in Guangzhou, Guangdong Province, with the license number: SYXK(Yue)2024-0136. All operations in this experiment complied with the ethical regulations of experimental animals. During the experiment, the mice were fed a basal diet at regular intervals and given free access to food and water; they were exposed to 12 h of light per day under constant temperature and humidity. After one week of adaptive feeding, the subsequent experimental operations were initiated.

[0077] 3. Animal grouping (8 mice / group)

[0078] Mice in the blank control group (C) were given a basal diet and equal amounts of pure water throughout the 1st to 12th days, and intragastrically administered an equal volume of 0.5% CMC-Na solution from the 13th to 19th days.

[0079] Mice in the model control group (M) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered an equal volume of 0.5% CMC-Na solution from the 13th to 19th days.

[0080] Mice in the sulfasalazine group (S) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered 100 mg / kg of sulfasalazine from the 13th to 19th days.

[0081] Mice in the low-dose echinacea-derived alkylamide group (AL) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered 2 mg / kg of echinacea-derived alkylamide from the 13th to 19th days.

[0082] Mice in the high-dose echinacea-derived alkylamide group (AH) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered 8 mg / kg of echinacea-derived alkylamide from the 13th to 19th days.

[0083] Mice in the low-dose echinacea-derived alkylamide liposome group (ALL) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered 2 mg / kg of echinacea-derived alkylamide liposome from the 13th to 19th days.

[0084] Mice in the high-dose echinacea-derived alkylamide liposome group (ALH) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered 8 mg / kg of echinacea-derived alkylamide liposome from the 13th to 19th days.

[0085] Mice in the folic acid blank liposome group (FL group) were given a basal diet, and were fed with 3% DSS in drinking water from the 1st to 12th days, and intragastrically administered 8 mg / kg of folic acid blank liposome from the 13th to 19th days.

[0086] Mice in the low-dose group of folic acid-echinacea alkylamide liposomes (FALL group) were fed a basal diet, and then given 3% DSS in drinking water from day 1 to day 12. From day 13 to day 19, they were given 2 mg / kg of folic acid-echinacea alkylamide liposomes by gavage.

[0087] Mice in the high-dose group of folic acid-echinacea-derived alkylamide liposomes (FALH group) were fed a basal diet, and then given 3% DSS in drinking water from day 1 to day 12. From day 13 to day 19, they were given 8 mg / kg of folic acid-echinacea-derived alkylamide liposomes by gavage.

[0088] During the experiment, each drug group was dissolved in 0.5% CMC-Na solution to prepare a liposome solution for gavage.

[0089] Example 1: Extraction and purification of echinacea-derived alkylamide compounds

[0090] 1. Experimental Methods:

[0091] See the operation process Figure 1 Weigh 2.5 kg of Echinacea root powder, soak and mix it in methanol, sonicate for 20 min, and then extract it three times at room temperature (8, 4 and 4 days respectively). Filter, collect and combine the methanol extracts, and concentrate them under reduced pressure at 50℃ to obtain crude extract of Echinacea root methanol extract.

[0092] The crude extract was vortexed with pure water and extracted three times with an equal volume of petroleum ether (boiling point 60–90 °C). The upper extract was collected, concentrated under reduced pressure at 50 °C to obtain the petroleum ether extract, which was then freeze-dried and weighed for later use.

[0093] The crude extract was further purified by silica gel column chromatography.

[0094] (1) Preparation of pre-column-passed samples

[0095] Weigh an appropriate amount of petroleum ether extract, dissolve and mix it with petroleum ether, add 8 times its weight of 200-300 mesh silica gel powder, mix well, concentrate under reduced pressure at 50°C and evaporate the solvent. This is the dry silica gel sample preparation method, and the column-passed sample is obtained.

[0096] (2) Packing of silica gel chromatography column

[0097] Select a suitable chromatography column and pre-wash the column thoroughly with n-hexane. Weigh 200-300 mesh silica gel powder according to a certain ratio (30-50 times the sample weight), mix it with n-hexane, use ultrasound to remove air bubbles, and then fill the column. Gently tap the column to remove air bubbles and fully compact the silica gel column for later use.

[0098] (3) Silica gel column chromatography elution and thin-layer chromatography analysis

[0099] The petroleum ether extract of Echinacea root was eluted using hexane:ethyl acetate as the eluent in silica gel column chromatography. The hexane:ethyl acetate ratio was continuously adjusted (9:1→5:1→3:1, v / v), and the eluent was collected based on the eluted bands. The collected eluent was analyzed by thin-layer chromatography (TLC) with hexane:ethyl acetate (3:1, v / v) as the developing solvent. The results were analyzed using a UV analyzer and compared with the standard. The eluent with parallel spots at 254 nm was collected, concentrated under reduced pressure (50℃), and the solvent was evaporated to obtain the target substance: Echinacea-derived alkylamide.

[0100] 2. Experimental Results

[0101] In this experiment, 2.5 kg of Echinacea root powder was extracted with methanol at room temperature. The methanol extracts were combined and concentrated by rotary evaporation under reduced pressure at 50 °C, yielding a crude methanol extract weighing 112.4 g. The crude extract was mixed with pure water and extracted three times with an equal volume of petroleum ether. The upper petroleum ether extract layer was collected, and the petroleum ether extracts were combined and concentrated by rotary evaporation under reduced pressure at 50 °C to obtain the petroleum ether extract. After freeze-drying, the extract weighed 21.2 g.

[0102] The crude extract was further purified by silica gel column chromatography. The eluent was collected according to the eluted bands and identified by TLC with standards. An eluent showing a color spot parallel to the standard solutions of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide indicated the presence of these substances. This demonstrates the successful extraction and purification of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramides from Echinacea root. The eluents containing these substances were combined and concentrated to obtain the purified alkylamide (i.e., Echinacea-derived alkylamide), which was then freeze-dried and stored at -20°C.

[0103] Example 2 Qualitative analysis of echinacea-derived alkylamide compounds

[0104] 1. Experimental Methods

[0105] 1.1 High Performance Liquid Chromatography Analysis

[0106] Chromatographic analysis was performed using COSMOSIL 5C. 18 -MS-II column (4.6×250mm), mobile phase is methanol (A)-pure water (B) (0~22min, 80% methanol), total flow rate is 0.8mL / min, injection is 10μL, detection wavelength is 254nm, column temperature is 35℃.

[0107] Preparation of reference solution: Dilute the mixture of dodecano-2E, 4E, 8Z, 10E / Z-tetraenoic acid isobutyramide with methanol and bring the volume to 10 mL to prepare a 1 mg / mL reference stock solution.

[0108] Preparation of alkylamide purified sample solution: Weigh about 10 mg of alkylamide purified sample, dissolve it in methanol and dilute to 10 mL to prepare alkylamide purified sample solution.

[0109] Preparation of petroleum ether extract test solution: Weigh about 10 mg of petroleum ether extract sample, dissolve it in methanol and dilute to 10 mL to prepare petroleum ether extract test solution.

[0110] According to the chromatographic conditions, 10 μL of the reference solution, the alkylamide purified test solution, and the petroleum ether extract test solution were injected for high performance liquid chromatography (HPLC) analysis.

[0111] (1) Examination of linearity: The reference stock solution was accurately pipetted and gradually diluted with methanol to 100, 50, 25, 10 and 5 μg / mL, and injected 3 times respectively. The determination was carried out under the chromatographic conditions of Example 1, and a standard curve was plotted based on the corresponding peak area.

[0112] (2) Precision test: The reference solution was precisely pipetted 6 times according to the above chromatographic conditions, the peak area was recorded and the relative standard deviation (RSD) value was calculated.

[0113] (3) Repeatability test: Prepare 6 parallel purified test solutions using the method of test solution preparation, inject 10 μL of each solution, record the peak area, and determine the RSD value.

[0114] (4) Stability test:

[0115] Preparation of the test solution: Prepare the purified test solution, place it at room temperature for 0, 1, 2, 4, 8, 12 and 24 h, inject 10 μL of each solution, record the peak area and determine the RSD value.

[0116] (5) Spike recovery test

[0117] Nine portions of alkylamide purified sample with known content were accurately weighed. Reference standards were added to the alkylamide purified sample at 80%, 100%, and 120% of the known contents of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide, respectively, to prepare alkylamide purified sample solutions. The contents of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide were determined according to the above chromatographic conditions, and the RSD values ​​were calculated.

[0118]

[0119] 1.2 Qualitative analysis using liquid chromatography-mass spectrometry

[0120] The purified sample was subjected to qualitative analysis by high-resolution liquid chromatography-high-resolution mass spectrometry (Q-TOF). Liquid chromatography conditions: Agilent SB-AqC18 reversed-phase column (2.1 × 100 mm, 1.8 μm), injection volume 5 μL, mobile phase 60% acetonitrile:40% water (containing 0.2% formic acid), column temperature 40℃, detection wavelength 254 nm. Mass spectrometry conditions: Dual AJS ESI ionization source, positive ion mode, mass-to-charge ratio (m / z) range 200–1700, scan rate 2.00 spectra / sec, VCap 4000 V, nozzle voltage 1000 V.

[0121] 2. Experimental Results

[0122] 2.1 Results of High Performance Liquid Chromatography (HPLC)

[0123] The results were obtained after HPLC analysis. Figure 2 The reference standards for dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide eluted at approximately 10 min (9.960 min). Figure 2 (a) in the middle, while petroleum ether extract ( Figure 2 (b) of the alkylamide purified product ( Figure 2 (c) also showed obvious peaks at around 10 min (9.974 min, 9.972 min), consistent with the reference standard, indicating the presence of isobutyramides, specifically dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid. Furthermore, the purified alkylamide showed significantly fewer peaks than the petroleum ether extract, and its baseline was flatter, indicating successful purification of alkylamide components, represented by dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramides, by silica gel column chromatography. However, the purified alkylamide also contained three distinct absorption peaks with good separation, suggesting substances other than dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramides, requiring further qualitative analysis.

[0124] The linear regression results obtained from the linear relationship examination showed that the linear regression equations for the dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide reference standards were y = 76927x + 45774, R0 2 =0.9997, indicating that the linear relationship of the dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide reference standards is good in the concentration range of 5–100 μg / mL.

[0125] The precision test results showed that the RSD value of the precision test was 1.35%, indicating that the instrument has good precision.

[0126] The repeatability test results showed an RSD value of 1.54%, indicating that the method has good repeatability.

[0127] The stability test results showed an RSD value of 1.80%, indicating that the sample solution has good stability.

[0128] The spiked recoveries are shown in Table 1 below. The average recoveries of isobutyramides of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid were 105.62%, with an RSD of 4.72%. This indicates that the method has good accuracy.

[0129] Table 1. Spike Recovery Results

[0130]

[0131] 2.2 Qualitative Analysis Results by Liquid Chromatography-Mass Spectrometry

[0132] The results are as follows Figure 3 As shown, in positive ion mode, six distinct components were collected by liquid chromatography-mass spectrometry (LC-MS) and analyzed by mass spectrometry (MS), with results shown in Table 2. Because the chromatographic conditions used in LC-MS Q-TOF analysis differed from those used in HPLC, the sample peak detected at 254 nm did not perfectly align with the retention time. Therefore, based on molecular weight analysis and literature comparison (Zhang Jing, 2012; Chen et al., 2005), it was inferred that compound 1 in the purified alkylamide sample detected by LC-MS Q-TOF was likely undecano-2E,4Z-diene-8,10-diyneic acid isobutyramide (252.1363 [M+Na]). + 459.3015[2M+H] + 481.2833[2M+Na] + Compound 2 may be dodecacarbon-2Z,4E,10Z-triene-8-acetylic acid isobutyramide (266.1520[M+Na)). + 487.3329 [2M+H] + 509.3151[2M+Na] +Compound 5 may be dodecano-2E, 4E, 8Z, 10E / Z-tetraenoic acid isobutyramide (248.2014[M+H)). + 495.3956 [2M+H] + 517.3770[2M+Na] + ()( Figure 4 ).

[0133] Table 2. Compound analysis results of purified alkylamide products using liquid chromatography-mass spectrometry.

[0134]

[0135] Example 3 Characterization of folic acid-echinacea-derived alkylamide liposomes

[0136] 1. Experimental Methods

[0137] Folic acid-echinacea-derived alkylamide liposome powder was evenly spread on conductive adhesive and mounted on a conductive stage. After being coated with gold using a vacuum coating instrument, its morphology was observed under a field emission scanning electron microscope at 24,927x and 79,965x magnification, respectively.

[0138] Weigh an appropriate amount of phosphotungstic acid hydrate and mix it with pure water to prepare a 2% phosphotungstic acid staining solution as a negative staining agent. Add an appropriate amount of sodium hydroxide solution to adjust the pH value of the phosphotungstic acid staining solution to 6.8-7.4 to ensure that the liposome morphology is not damaged during negative staining. Add the folic acid-echinacea-derived alkylamide liposome suspension to a copper grid and let it stand for 2-3 minutes until the sample solution is adsorbed onto the copper grid. Use filter paper to absorb the excess solution, add the staining solution to the copper grid, stain for 20-30 seconds, absorb the excess staining solution with filter paper, wash twice with pure water, and finally blot dry. After air drying, observe the morphology using a transmission electron microscope.

[0139] After diluting the folic acid-echinacea-derived alkylamide liposome suspension with PBS solution, the particle size, zeta potential, and polydispersity index (PDI) of the folic acid-alkylamide liposomes were determined using a nano-laser particle size analyzer.

[0140] 2. Experimental Results

[0141] like Figure 5 As shown, under a scanning electron microscope, most of them are spherical with smooth surfaces, relatively uniform size, small particle size, and good uniformity. Figure 6 As shown, folic acid-echinacea-derived alkylamide liposomes were observed to be uniformly distributed under transmission electron microscopy; most were spherical and intact in shape; the particle size was small, mostly within 200 nm, and the particle size uniformity was good. Figure 7As shown, the particle size of folic acid-echinacea-derived alkylamide liposomes is mainly concentrated between 100 and 200 nm, with a relatively concentrated particle size distribution. The average particle size of the liposomes was measured to be 122.7 ± 0.4 nm, the average potential was -2.87 mV, and the PDI was 0.114.

[0142] Example 4: HPLC analysis of folic acid-echinacea-derived alkylamide liposomes

[0143] 1. Experimental Methods

[0144] The presence of the obtained alkylamide compound in the prepared folic acid-echinacea-derived alkylamide liposome sample was determined by HPLC. The chromatographic conditions were the same as in Example 2.

[0145] Preparation of folic acid-echinacea-derived alkylamide liposome solution: Transfer 1 mL of folic acid-echinacea-derived alkylamide liposome suspension to methanol for demulsification, sonicate for 10 min, and then adjust the volume to 10 mL to prepare the folic acid-echinacea-derived alkylamide liposome test solution.

[0146] Methodological investigations were conducted on dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramides, using the same methods as in Example 2.

[0147] 2. Experimental Results

[0148] The results are as follows Figure 8 As shown, the dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide reference standards showed peaks at around 11 min, and the folic acid-echinacea-derived alkylamide liposome test sample also showed obvious peaks at the corresponding time, consistent with the reference standards. This indicates that the prepared liposome samples contained purified alkylamides, mainly dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide.

[0149] Example 5: Stability and release rate analysis of folic acid-echinacea-derived alkylamide liposomes

[0150] 1. Experimental Methods

[0151] Folic acid-echinacea-derived alkylamide liposome suspensions were placed at 4℃ and 25℃, and the particle size and potential of the folic acid-echinacea-derived alkylamide liposomes were measured at 0, 5, 10, 15, 20, 25 and 30 days.

[0152] An appropriate amount of folic acid-echinacea-derived alkylamide liposome suspension was placed in a dialysis bag (MWCO: 5000 Da) and then placed in 50 mL of PBS solution (pH = 7.4) and acetic acid solution (pH = 4.4) containing 0.5% Tween 80, respectively. The mixture was shaken at a constant temperature (37℃, 100 r / min). At 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h, 1 mL of release medium was collected and the corresponding volume of 0.5% Tween 80 solution was added. The content of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide in the released samples at these time points was determined by HPLC, and the cumulative release rate was calculated.

[0153]

[0154] Note: Ci: Concentration of the release medium at the i-th sampling, V: Volume of the release medium at each sampling, Ct: Concentration of the release medium at the current time point; V0: Total volume of the release medium, m: Total amount of dodecano-2E, 4E, 8Z, and 10E / Z-tetraenoic acid isobutyramide in the liposome solution, mg.

[0155] 2. Experimental Results

[0156] The results are as follows Figure 9 As shown, the particle size and potential of folic acid-echinacea-derived alkylamide liposome suspension stored at 4℃ for 5, 10, 15, 20, 25, and 30 days showed some changes compared to 0 days, but the changes were small. For example, the particle size increased, but the change was within 5 nm. Therefore, it is considered that the folic acid-echinacea-derived alkylamide liposome suspension still has good stability after 30 days at 4℃. At 25℃ for 5, 10, 15, 20, 25, and 30 days, the particle size decreased compared to 0 days, but the change was within 10 nm. The change in potential was larger than at 4℃. It is considered that the folic acid-echinacea-derived alkylamide liposome suspension still has some stability after 30 days at 25℃. Overall, the folic acid-echinacea-derived alkylamide liposome suspension is considered more stable at 4℃ than at 25℃, and is therefore more suitable for storage.

[0157] The results are as follows Figure 10 As shown, the drug release process of folic acid-echinacea-derived alkylamide liposomes exhibits a gradual increase over time. The drug release rate continuously rises from 0 to 8 hours, then increases slowly from 8 to 12 hours, reaching a peak around 12 hours. This indicates that folic acid-echinacea-derived alkylamide liposomes possess good physical stability and exhibit excellent sustained-release properties, with the drug release rate reaching its highest point around 12 hours.

[0158] Example 6: Growth status and DAI scores of mice in each group

[0159] 1. Experimental Methods

[0160] Based on the human-to-mouse body surface area equivalent dose ratio of 0.0025, the low-dose and high-dose groups of folic acid-echinacea alkylamide liposomes were set at 2 and 8 mg / kg, respectively. The experiment was divided into 10 groups, with 8 mice in each group: blank control group (C), model control group (M), sulfasalazine group (S), low-dose echinacea alkylamide group (AL), high-dose echinacea alkylamide group (AH), low-dose echinacea alkylamide liposome group (ALL), high-dose echinacea alkylamide liposome group (ALH), folic acid blank liposome group (FL), low-dose folic acid-echinacea alkylamide liposome group (FALL), and high-dose folic acid-echinacea alkylamide liposome group (FALH). Five-week-old male KM mice weighing 21–33 g were used. Mice were fed 3% DSS in drinking water for 12 days, followed by gavage administration of the drugs once daily for 7 days. Mice were weighed, and daily weight changes, fecal viscosity, and fecal occult blood were recorded. The DAI score for each group of mice was calculated. The detailed scoring rules are shown in Table 3. DAI score = (percentage weight loss score + fecal viscosity score + fecal occult blood score) / 3.

[0161] Table 3. Detailed criteria for DAI scoring in each group of mice.

[0162]

[0163] 2. Experimental Results

[0164] Changes in body weight of mice in each group during the experiment are as follows: Figure 11 As shown, the body weight of mice in each modeling group was significantly different from that of mice in group C during the modeling period (0-12d) (P<0.05). The body weight gradually decreased from day 6, reaching its lowest point between days 10 and 12. On day 12 of the experiment, each drug treatment group began gavage treatment. During the treatment period, the body weight of mice in each drug treatment group gradually increased, but there was no significant difference from that of mice in group M (P>0.05).

[0165] Mouse DAI score during the experiment: Figure 12 As shown, compared with the blank control group, the DAI scores of mice in all groups were significantly increased after the onset of the model (P<0.05), reaching their maximum value during days 10-12 of the experiment. After drug treatment began on day 12 of the experiment, the DAI scores of mice in all drug-treated groups gradually decreased, reaching their lowest point during days 16-18 of the experiment. During the treatment period, compared with group M, except for group S, the DAI scores of other drug-treated groups were significantly reduced (P<0.05).

[0166] Example 7: Changes in colon length and organ index in mice of different groups

[0167] 1. Experimental Methods

[0168] The experimental groups were as described in Example 6. 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 colonic segment was rinsed with PBS solution, laid flat, and the colonic length was measured. The heart, liver, spleen, lungs, kidneys, and cecum were harvested, weighed, and recorded. Based on the mouse body weight, organ weight, and colonic length recorded at the time of sample collection, the organ index of each group of mice was calculated.

[0169] Organ index = organ weight (g) / body weight (g) × 100%.

[0170] 2. Experimental Results

[0171] The results are as follows Figure 13 As shown in the figure. After modeling, compared with group C, the colon length of group M was significantly shortened (P<0.01). After treatment with the drug, the colon length of mice in each group recovered to some extent. Among them, the colon length of mice in group S increased significantly (P<0.01), the colon length of mice in groups AL and ALH increased significantly (P<0.05), and the colon length of mice in group FALH recovered significantly but there was no significant difference (P>0.05).

[0172] The results of organ index changes are shown in Table 4. Compared with group C, the liver, spleen, and colon indices of mice in group M were significantly increased (P<0.05), while the lung and kidney indices were significantly decreased (P<0.05), and the heart index showed no significant difference (P>0.05). Compared with group M, the heart, lung, and kidney indices of group S were significantly increased (P<0.05), and the lung and kidney indices of group FALH were significantly increased (P<0.05). Most drug-treated groups showed a decreasing trend in heart, liver, spleen, and colon indices, but none of these differences were significant (P>0.05).

[0173] Table 4. Organ indices of mice in each group (g, X±SD, n=5)

[0174]

[0175]

[0176] Note: If the superscript letters of the values ​​in the table are the same, it means there is no significant difference between the groups (P > 0.05). If the letters are different, it means there is a significant difference between the groups (P < 0.05). "a" represents the maximum value, and "b" and "c" represent decreasing values ​​in that order.

[0177] Example 8: Observation of histopathological sections of mouse colon tissue

[0178] 1. Experimental Methods

[0179] The experimental groups were as described in Example 6. Small segments of colon tissue were excised from each group of mice, fixed in 4% paraformaldehyde solution for one week, dehydrated by gradient elution with ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The pathological damage of the colon tissue in each group of mice was observed after panoramic scanning of the sections.

[0180] 2. Experimental Results

[0181] The results are as follows Figure 14 As shown, the colonic tissue structure in group C was intact, with neatly arranged crypts, no inflammatory cell infiltration, and no obvious pathological changes. In group M, the colonic crypt structure was destroyed, accompanied by abscesses, goblet cell depletion, and extensive inflammatory cell infiltration. In group S, the colonic crypt structure was intact, but with a small amount of inflammatory cell infiltration. In group AL, some crypt structures were lost, with mild crypt abscesses, a small amount of inflammatory cell infiltration, and goblet cell depletion; while in group AH, a small amount of inflammatory cell infiltration was observed in the colon, and the crypt structure was relatively intact. In group ALL, the colonic crypt structure was destroyed, with inflammatory cell infiltration and goblet cell depletion; in group ALH, the crypts were neatly arranged, and a small amount of inflammatory cell infiltration was observed. In group FL, some colonic crypt structures were destroyed and abscesses were present, with goblet cell depletion and extensive inflammatory cell infiltration. In group FALL, a few inflammatory cells were observed in the colon, but no obvious pathological abnormalities were seen; in group FALH, the colonic crypt structure was relatively intact, with some goblet cell depletion and no obvious inflammatory cell infiltration.

[0182] Example 9: Determination of the levels of inflammatory factors in the serum of mice in each group

[0183] 1. Experimental Methods

[0184] The experimental groups were as described in Example 6. After anesthetizing the mice, the eyeballs were removed and blood was collected in non-anticoagulated blood collection tubes. The plasma samples were centrifuged at 3500 rpm for 8 minutes, and the supernatant was collected. The levels of TNF-α, IL-6, IL-1β, IL-10, and COX2 in the serum of each group of mice were measured according to the instructions of the enzyme-linked immunosorbent assay (ELSIA) kit.

[0185] 2. Experimental Results

[0186] The results are as follows Figure 15As shown in the figure. Compared with group C, the serum levels of TNF-α, IL-1β, and COX2 in group M mice were significantly increased (P<0.01), the IL-6 level was significantly increased (P<0.05), and the IL-10 level was significantly decreased (P<0.001). Compared with group M, the serum levels of TNF-α, IL-1β, IL-6, and COX2 in group S mice were significantly decreased (P<0.05), significantly decreased (P<0.001), significantly decreased (P<0.01), and significantly increased (P<0.01) in group S mice. The serum levels of TNF-α, IL-1β, and IL-10 in group AL mice were significantly decreased (P<0.05), significantly decreased (P<0.01), and significantly increased (P<0.01) in group AL mice. The serum levels of IL-6 and IL-10 in group AH mice were significantly decreased (P<0.001) and significantly increased (P<0.01). 01); In the ALL group, the serum TNF-α level was significantly decreased (P<0.01), the IL-1β and IL-6 levels were extremely significantly decreased (P<0.001), the COX2 level was significantly decreased (P<0.01), and the IL-10 level was significantly increased (P<0.001); In the ALH group, the serum TNF-α, IL-1β, and COX2 levels were significantly decreased (P<0.05), the IL-6 level was extremely significantly decreased (P<0.001), and the IL-10 level was significantly increased (P<0.001); In the FALL and FALH groups, the serum TNF-α level was significantly decreased (P<0.05), the IL-1β and IL-6 levels were extremely significantly decreased (P<0.001), the COX2 level showed a decreasing trend but no significant difference (P>0.05), and the IL-10 level was significantly increased (P<0.001).

[0187] Example 10: Determination of mRNA expression levels of inflammatory factors in colon tissues of mice in each group

[0188] 1. Experimental Methods

[0189] The experimental groups were as described in Example 6. Colon tissue was homogenized, centrifuged, and the supernatant was collected. 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, IL-1β, iNOS, COX2, IL-10, CD86, CD206, and Arg-1 in the colon.

[0190] Table 5 Primer sequences

[0191]

[0192] 2. Experimental Results

[0193] The results are as follows Figure 16 As shown, compared with the blank control group, the mRNA expression levels of TNF-α, IL-6, IL-1β, iNOS, COX2 and CD86 in the colon of mice in the model control group were significantly increased (P < 0.001); compared with the model control group, the mRNA expression levels of TNF-α, IL-6, IL-1β, iNOS, COX2 and CD86 in the colon tissue of mice in the ALL, ALH, FALL and FALH groups were all significantly decreased (P < 0.001).

[0194] Compared with the blank control group, the mRNA expression levels of IL-10 and Arg-1 in the colon tissue of mice in the model control group showed an increasing trend but no significant difference (P>0.05), while the mRNA expression level of CD206 was significantly decreased (P<0.05). Compared with the model control group, the mRNA expression levels of IL-10, Arg-1, and CD206 in the colon tissue of mice in each drug-treated group were significantly increased (P<0.001, P<0.05, P<0.05).

[0195] Example 11: Determination of mRNA expression levels of signaling pathways in the colon of mice in each group

[0196] 1. Experimental Methods

[0197] The experimental groups were as described in Example 6. Colon tissue was homogenized, centrifuged, and the supernatant was collected. 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 6). 2 -ΔΔCT The relative quantitative analysis was performed to determine the mRNA expression levels of Nrf2, HO-1, NF-κB, STAT3, and JAK2 in the colon.

[0198] Table 6 Primer sequences

[0199]

[0200]

[0201] 2. Experimental Results

[0202] The results are as follows Figure 17As shown, compared with the blank control group, the mRNA expression levels of NF-κB, JAK2, and STAT3 in the colon tissue of mice in the model control group were significantly increased (P<0.001). Compared with the model control group, the mRNA expression levels of NF-κB and JAK2 in the colon tissue of mice in the ALL and FALH groups were significantly decreased (P<0.01). The mRNA expression level of JAK2 in the colon tissue of mice in the ALH and FL groups was significantly decreased (P<0.001); the mRNA expression level of STAT3 in the colon tissue of mice in the ALH and FALH groups was significantly decreased (P<0.05).

[0203] Compared with the blank control group, the mRNA expression level of Nrf2 in the colon tissue of mice in the model control group was significantly increased (P<0.01). Compared with the model control group, the mRNA expression level of Nrf2 in the colon tissue of mice in the ALH group and FALH group was significantly increased (P<0.01).

[0204] Compared with the blank control group, the mRNA expression level of HO-1 in the colon tissue of mice in the model control group showed an increasing trend but no significant difference (P>0.05). Compared with the model control group, the mRNA expression level of HO-1 in the colon tissue of mice in each drug treatment group was significantly increased (P<0.05), among which the mRNA expression level of HO-1 in the colon tissue of mice in the AL group, AH group, ALH group and FALH group was significantly increased (P<0.001).

[0205] In summary, mice with ulcerative colitis treated with folic acid-echinacea-derived alkylamide liposomes showed superior performance compared to other single-drug groups in terms of weight change, colon length, DAI score, organ index, colonic histopathological damage, and anti-inflammatory effects. This indicates that folic acid-echinacea-derived alkylamide liposomes have a good therapeutic effect on DSS-induced ulcerative colitis, and that folic acid and echinacea-derived alkylamides have a certain synergistic effect in alleviating inflammation in mice with ulcerative colitis. The main components of folic acid-echinacea-derived alkylamide 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-echinacea-derived alkylamide liposomes can provide a new approach for the treatment of ulcerative colitis in livestock farming.

[0206] Furthermore, through multiple experiments, the inventors discovered that folic acid-echinacea-derived alkylamide liposomes prepared by weighing the raw materials in a mass ratio of egg yolk lecithin:cholesterol:echinacea-derived alkylamide:folic acid of 4–8:2–5:1–2:1–2 resulted in liposomes with a high encapsulation efficiency, with a mass ratio of 6:3:1:1 being the optimal ratio. Dosage screening revealed that folic acid-echinacea-derived alkylamide liposomes at doses of 2–8 mg / kg were effective, with an oral administration of 8 mg / kg maximizing the drug's cost-effectiveness.

[0207] 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.

[0208] 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. The use of a folic acid-echinacea-derived alkylamide liposome in the preparation of a medicament for treating ulcerative colitis, characterized in that: The folic acid-echinacea-derived alkylamide liposomes are composed of egg yolk lecithin, cholesterol, echinacea-derived alkylamide, phospholipid-polyethylene glycol-folic acid, and are prepared by ethanol injection method.

2. The application according to claim 1, characterized in that: The mass ratio of egg yolk lecithin, cholesterol, echinacea-derived alkylamide, phospholipid-polyethylene glycol-folic acid is 4-8:2-5:1-2:1-2.

3. The application according to claim 2, characterized in that: The mass ratio of egg yolk lecithin, cholesterol, echinacea-derived alkylamide, phospholipid-polyethylene glycol-folic acid is 6:3:1:

1.

4. The application according to any one of claims 1 to 3, characterized in that: The ulcerative colitis mentioned is ulcerative colitis induced by DSS as a pathogenic factor; The drug is administered via gavage.

5. The application according to any one of claims 1 to 3, characterized in that: The dosage of the drug for treating ulcerative colitis is 2–8 mg / kg per individual.

6. The application according to any one of claims 1 to 3, characterized in that: For at least one of the following applications: (1) The treatment for ulcerative colitis involves increasing body weight and decreasing DAI score; (2) The treatment for ulcerative colitis involves increasing colon length; decreasing heart, liver, spleen, and colon indices; and increasing lung and kidney indices. (3) The treatment for ulcerative colitis aims to improve the degree of pathological damage and inflammatory response of colonic tissue; (4) The treatment for ulcerative colitis involves downregulating the levels of inflammatory factors TNF-α, IL-1β, IL-6, and COX2 in the serum, and upregulating the level of the anti-inflammatory factor IL-10 in the serum. (5) The treatment for ulcerative colitis involves downregulating the levels of inflammatory factors TNF-α, IL-6, IL-1β, iNOS, COX2 and CD86 in the colon; and upregulating the levels of anti-inflammatory factors IL-10, Arg-1 and CD206 in the colon. (6) The treatment of ulcerative colitis involves downregulating the mRNA expression levels of related signaling pathways NF-κB, JAK2 and STAT3 in the colon, and upregulating the mRNA expression levels of related signaling pathways Nrf2 and HO-1 in the colon.

7. The method for preparing folic acid-echinacea-derived alkylamide liposomes according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Echinacea root powder was soaked and extracted with methanol, the methanol extract was collected, and the extract was concentrated under reduced pressure to obtain crude extract of Echinacea root methanol extract. S2. Mix the crude extract with pure water by vortexing, extract three times with an equal volume of petroleum ether, collect the upper extract, concentrate under reduced pressure to obtain the petroleum ether extract, and freeze-dry. S3. Silica gel column chromatography, collect the eluent for thin-layer chromatography analysis, and use an ultraviolet analyzer to detect the eluent that has parallel colored spots with the standard at a wavelength of 254 nm. Then, concentrate under reduced pressure and evaporate the solvent to obtain the purified alkylamide, namely echinacea-derived alkylamide. S4. Dissolve phospholipid-polyethylene glycol-folic acid in a small amount of dichloromethane, evaporate to dryness to form a film, dissolve in PBS and mix thoroughly to obtain the aqueous phase; weigh egg yolk lecithin, cholesterol, and echinacea-derived alkylamide and dissolve in anhydrous ethanol, mix thoroughly to obtain the oil phase; S5. Slowly inject the oil phase into the aqueous phase and continue stirring to evaporate the ethanol and remove most of the organic solvent; S6. Continue stirring to evaporate and remove the remaining organic solvent, so that the oil phase and water phase are fully mixed; S7. After cooling, sonication, filtration, and freeze-drying, folic acid-echinacea-derived alkylamide liposome powder is obtained.

8. The preparation method according to claim 7, characterized in that: In step S3, the eluent used for elution is a gradient mixture of n-hexane and ethyl acetate. In step S5, the continuous stirring conditions are 400-600 r / min for 10-20 min; In step S6, the stirring speed is 400-600 r / min.

9. The preparation method according to claim 7, characterized in that: In step S7, the ultrasound duration is 5–7 minutes; The filtration process uses a 0.22μm filter membrane.

10. A folic acid-echinacea-derived alkylamide liposome, characterized in that: It is prepared by the preparation method according to any one of claims 7 to 9.

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