Preparation method of a compound regulator containing omega-3 fatty acid-lipoxin-phospholipid for enteritis of pelteobagrus fulvidraco

Abstract

The present application relates to the technical field of aquatic disease prevention and control, and particularly relates to a preparation method of a compound regulator for enteron inflammation of Pelteobagrus fulvidraco containing omega-3 fatty acid-lipoxin-phospholipid. The functional components of the compound regulator include eicosapentaenoic acid 20-30 parts, lipoxin B4 0.1-0.5 parts, phosphatidylserine 8-12 parts, dehydroabietic acid 1-2 parts, sophorobiose 10-18 parts and ethoxyquinoline 0.01-0.03 parts. The product integrates the functions of antibiosis, inflammation subsiding and tissue repair through a multi-target synergistic mechanism, and builds a complete inflammation regulation pathway. Meanwhile, the active ingredients are effectively protected by means of nano-liposome dosage form technology. The product realizes the antibiosis function by using natural ingredients. The technical scheme can solve the technical problems of low antibiosis efficiency, single mechanism and insufficient mucosal repair capacity of the existing prevention and control technology for enteron inflammation of Pelteobagrus fulvidraco, and has an ideal application prospect.

Description

Technical Field

[0001] This invention relates to the field of aquatic disease prevention and control technology, specifically to a method for preparing a compound regulatory agent for intestinal inflammation in yellow catfish containing ω-3 fatty acids, lipoxygen, and phospholipids. Background Technology

[0002] Yellow catfish ( Pelteobagrus fulvidraco As an important freshwater aquaculture fish in my country, the yellow catfish is highly favored by consumers due to its tender flesh, rich nutrition, and lack of intramuscular bones, leading to a continuous increase in its farming scale and density in recent years. Under intensive farming models, the intestinal health of the yellow catfish has become a key factor restricting its farming efficiency. The intestine is not only the core organ for the digestion and absorption of nutrients but also an important physical barrier against the invasion of external pathogens. The intestinal mucosa of the yellow catfish is relatively fragile, exhibiting low tolerance to pathogens and stress factors in the farming environment, making it highly susceptible to intestinal inflammation and causing significant economic losses to the aquaculture industry.

[0003] The causes of intestinal inflammation in yellow catfish are multifaceted, with pathogenic microbial infection and environmental stress being the two core factors. At the pathogen level, opportunistic pathogens such as Vibrio (e.g., Vibrio mimicus) and Aeromonas hydrophila are the main causative agents. These bacteria can damage the mucosal barrier by adhering to intestinal epithelial cells, leading to intestinal congestion, edema, and inflammatory cell infiltration. At the environmental level, stress factors such as fluctuations in dissolved oxygen in the aquaculture water, sudden temperature changes, and nutritional imbalances in the feed can further reduce the immunity of yellow catfish, exacerbating the occurrence and development of intestinal inflammation.

[0004] In the traditional treatment of intestinal inflammation in yellow catfish, antibiotics are widely used due to their rapid onset and clear antibacterial effects. However, the long-term irrational use of antibiotics has led to a series of prominent problems: on the one hand, pathogenic microorganisms easily develop drug resistance, resulting in a gradual decline in the effectiveness of antibiotic treatment, and even a situation where no drugs are available; on the other hand, antibiotic residues can enter the human body through the food chain, endangering public health, and at the same time causing serious damage to the microecological balance of aquaculture water bodies. Therefore, developing safe, efficient, and environmentally friendly antibiotic-free intestinal inflammation prevention and control solutions has become an inevitable requirement for the industry.

[0005] Currently, some antibiotic-based technologies for the prevention and control of intestinal inflammation have emerged in the aquaculture industry. However, there are still significant technical deficiencies in specialized regulatory agents for intestinal inflammation in yellow catfish, making it difficult to meet actual aquaculture needs. These deficiencies manifest in the following three aspects:

[0006] (1) Low efficacy of natural antibacterial ingredients: Although commonly used single plant essential oils in existing technologies (such as thymol, cinnamaldehyde, etc.) have certain natural antibacterial activities, they generally have a narrow antibacterial spectrum. Taking the inhibitory effect on Vibrio, a common pathogen causing intestinal inflammation in yellow catfish, as an example, the minimum inhibitory concentration (MIC) of such single ingredients is usually ≥100μg / mL, which is far higher than the effective concentration range that is easy to add in actual aquaculture. Therefore, it is difficult to quickly control the spread of pathogens in the early stage of inflammation and to curb the spread of the disease.

[0007] (2) The linkage between inflammation and oxidative stress is not blocked: After intestinal infection in yellow catfish, pathogen stimulation will trigger the explosive production of reactive oxygen species (ROS) in intestinal epithelial cells. Excessive ROS will not only directly damage intestinal mucosal cells, but also activate inflammatory signaling pathways, promoting the secretion of large amounts of inflammatory factors such as interleukin-6 (IL-6)-like factors and tumor necrosis factor. The release of inflammatory factors will further aggravate the generation of ROS, forming a positive feedback loop of oxidative stress and inflammation, which will accelerate intestinal damage. Most existing regulatory agents only target a single link in the inflammatory pathway or oxidative stress pathway, and fail to fundamentally block the linkage mechanism between the two. In particular, the inhibitory effect on key pro-inflammatory factors such as interleukin-6 (IL-6) is limited, resulting in unsatisfactory inflammation control.

[0008] (3) Insufficient intestinal mucosal repair mechanism: The integrity of the intestinal mucosal barrier is the key to preventing the recurrence of intestinal inflammation in yellow catfish, and tight junction proteins (such as ZO-1 and Occludin proteins) are the core substances for maintaining tight junctions between intestinal epithelial cells and ensuring the function of the mucosal barrier. Existing antibiotic-free agents mostly focus on antibacterial or anti-inflammatory effects, and lack active ingredients that can directly promote the synthesis of tight junction proteins by intestinal epithelial cells of yellow catfish. This leads to slow intestinal mucosal repair. Even if the inflammation is relieved in the short term, it is easy to cause chronic inflammation to recur due to the weak mucosal barrier function, and it is impossible to achieve long-term regulation of intestinal health.

[0009] In summary, existing prevention and control technologies for intestinal inflammation in yellow catfish suffer from problems such as low antibacterial efficiency, single mechanism of action, and insufficient mucosal repair capacity. There is an urgent need to develop a compound regulatory agent that can simultaneously achieve highly efficient antibacterial activity, block the oxidative stress-inflammation linkage, and promote intestinal mucosal repair to meet the needs of the healthy development of the yellow catfish aquaculture industry. Summary of the Invention

[0010] The present invention aims to provide a compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygenin and phospholipids, in order to solve the technical problems of existing prevention and control technologies for intestinal inflammation of yellow catfish, such as low antibacterial efficiency, single mechanism of action and insufficient mucosal repair capacity.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A compound preparation for regulating intestinal inflammation in yellow catfish containing ω-3 fatty acids, lipoxygenase, and phospholipids, wherein the active ingredients, by weight, include: 20-30 parts eicosapentaenoic acid, 0.1-0.5 parts lipoxygenase B4, 8-12 parts phosphatidylserine, 1-2 parts dehydrorosinic acid, 10-18 parts sophorobiose, and 0.01-0.03 parts ethoxyquinoline.

[0013] In summary, the technical principle of this invention is as follows:

[0014] Furthermore, the phosphatidylserine is a 14:0 / 14:0 type phosphatidylserine.

[0015] Furthermore, a compound regulatory preparation for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygenase, and phospholipids, by weight, contains the following active ingredients: 25 parts eicosapentaenoic acid, 0.3 parts lipoxygenase B4, 10 parts phosphatidylserine, 1 part dehydrorosinic acid, 15 parts sophorobiose, and 0.02 parts ethoxyquinoline.

[0016] This technical solution also provides a method for preparing a compound regulatory agent for intestinal inflammation in yellow catfish containing ω-3 fatty acids, lipoxygenin, and phospholipids, including the following steps:

[0017] S1: Lipid membrane preparation

[0018] The lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, and dehydrorosinic acid were added to a chloroform-methanol mixed solvent to obtain a lipid solution; the lipid solution was subjected to rotary evaporation to obtain a lipid membrane loaded with the drug.

[0019] S2: Aqueous phase preparation

[0020] Sophora bisaccharide and ethoxyquinoline were added to a buffer solution and mixed to obtain an aqueous solution. The aqueous solution was preheated and then added to a container containing a lipid membrane loaded with the drug. The mixture was stirred until the lipid membrane was hydrated.

[0021] S3: Preparation of nanoliposome suspension

[0022] The hydrated lipid membrane forms a crude emulsion, which is then subjected to ultrasonic treatment and liposome extrusion to obtain a nanoliposome suspension.

[0023] S4: Freeze-drying molding

[0024] Mannitol and trehalose were added to the nanoliposome suspension, and after vacuum freeze-drying, a compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygen, and phospholipids was obtained.

[0025] Furthermore, in S1, the mass ratio of lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, and dehydrorosinic acid is 30-50: 20-30: 0.1-0.5: 8-12: 1-2.

[0026] Furthermore, in S1, the lipid carrier is composed of soybean lecithin and cholesterol in a mass ratio of 60-80:20-40;

[0027] The ratio of lipid carrier to chloroform-methanol mixed solvent is 40-60g: 200-400mL; the chloroform-methanol mixed solvent is formed by mixing chloroform and methanol in a volume ratio of 1-2: 1-2.

[0028] The rotary evaporation process is as follows: under reduced pressure at 60°C, the solvent is removed by rotary evaporation until a uniform lipid film is formed on the inner wall of the flask; nitrogen is purged for 30 min at 50°C and 0.2 MPa pressure, ensuring that the residual chloroform content is ≤50 ppm; then, the film is vacuum dried at 40°C for 2 h to remove the residual solvent and obtain the lipid film loaded with the drug.

[0029] Furthermore, in S2, the ratio of sophorobiose, ethoxyquinoline, and buffer solution is 10-18 g : 0.01-0.03 g : 300-500 mL;

[0030] After preheating the aqueous solution to 55°C, lipid membrane hydration was performed.

[0031] The buffer solution is a phosphate buffer.

[0032] Furthermore, in S3, the ultrasonic processing parameters are 200W power and 40kHz frequency, and the ultrasonic processing is performed for 15 minutes according to the procedure of 3 seconds of operation and 3 seconds of rest.

[0033] The liposome extrusion process used a polycarbonate membrane with a pore size of 100 nm and was filtered three times.

[0034] Furthermore, in S4, mannitol at a final concentration of 2-4% and trehalose at a final concentration of 1-3% are added to the nanoliposome suspension as freeze-drying protectants.

[0035] The vacuum freeze-drying procedure is as follows: pre-freeze at -40℃ for 2 hours; then dry at -40℃ and vacuum degree <10Pa for 12 hours, followed by heating to 25℃ at a rate of 0.5℃ / min; after the heating is completed, continue drying for 2 hours.

[0036] This technical solution also provides the application of a compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids in the preparation of drugs to alleviate intestinal inflammation of yellow catfish caused by Vibrio mimicry infection, or in the preparation of drugs to inhibit Vibrio mimicry infection.

[0037] The technical principle of this technical solution is as follows:

[0038] This invention achieves multi-target regulation of intestinal inflammation in yellow catfish by constructing a ternary synergistic system of ω-3 fatty acids, lipooxygen, and phospholipids, and combining it with nanoliposome delivery technology.

[0039] Dehydrorosin acid in the plant antibacterial complex can disrupt the lipid bilayer of bacterial cell membranes, with a minimum inhibitory concentration (MIC) of 15 μg / mL against Vibrio mimicry, directly inhibiting the proliferation of pathogens. Eicosapentaenoic acid (EPA) in the ω-3 fatty acid group reduces intestinal damage through a dual pathway of pathogen elimination and virulence blocking by inhibiting the Vibrio quorum sensing system and reducing the secretion of virulence factors such as hemolysin. The pro-inflammatory mediator lipoxygenin B4 specifically activates inflammatory pathway receptors, triggering the inflammatory resolution pathway, promoting macrophage clearance of apoptotic cells, and downregulating pro-inflammatory responses to achieve precise termination of inflammation and prevent prolonged inflammation. The 14:0 / 14:0 type phosphatidylserine (PS) in the phospholipid repair system can directly integrate into the intestinal mucosal cell membrane of yellow catfish, specifically repairing the tight junction protein ZO-1, reducing intestinal permeability, rebuilding the intestinal physical barrier, and enhancing the ability to resist pathogen invasion. Ethoxyquin can specifically scavenge hydroxyl radicals at the site of infection. Sophora bisaccharide (OH) protects intestinal cell DNA from oxidative damage; by regulating the pH of the intestinal mucus layer, it inhibits the binding of Vibrio mimicry surface adhesion receptors (such as fucose residues) to intestinal epithelial cells, while optimizing the intestinal microecological environment and helping to enhance barrier function.

[0040] By using nanoliposome encapsulation technology, various components can be stably coexisted and targetedly delivered, avoiding inactivation of components in the gastrointestinal environment and ensuring the synergistic effect of various mechanisms of action. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0042] Example 1

[0043] Table 1 shows the active ingredients, content, and range of each ingredient in a compound preparation for regulating intestinal inflammation in yellow catfish containing ω-3 fatty acids, lipoxygen, and phospholipids.

[0044] Table 1: Formula Ingredients and Proportions (Effective Ingredients)

[0045]

[0046]

[0047] Equation (1)

[0048] In addition to the aforementioned active ingredients, lipid carriers, aqueous solvents, and lyophilization protectants are also required. The raw materials for the lipid carrier include soybean lecithin (meeting the requirements for "soybean lecithin" in the 2025 edition of the Chinese Pharmacopoeia, Volume IV, CAS No.: 8030-76-0) and cholesterol (CAS No.: 57-88-5); the aqueous solvent can be phosphate-buffered saline (PBS); and the lyophilization protectants include mannitol and trehalose.

[0049] The compound regulatory preparation shall be formulated according to the following method:

[0050] Step 1: Lipid membrane preparation

[0051] A mixture of lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, and dehydrorosinic acid in a mass ratio of 30-50:20-30:0.1-0.5:8-12:1-2 is added to a chloroform-methanol mixed solvent. Preferably, the mass ratio of lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, and dehydrorosinic acid is 40:25:0.3:10:1. The lipid carrier is composed of soybean lecithin and cholesterol in a mass ratio of 60-80:20-40 (preferably 70:30); the ratio of lipid carrier to chloroform-methanol mixed solvent is 40-60g:200-400mL (preferably 50g:300mL), and the chloroform-methanol mixed solvent is formed by mixing chloroform and methanol in a volume ratio of 1-2:1-2 (preferably 2:1). The mixture is magnetically stirred in a 37°C water bath until completely dissolved, forming a clear lipid solution.

[0052] The lipid solution was transferred to a rotary evaporator and the solvent was removed by rotary evaporation at 60°C under reduced pressure (≤50 mbar) until a uniform lipid film formed on the inner wall of the flask. The flask was then purged with nitrogen (50°C, 0.2 MPa pressure, for approximately 30 min) to ensure that the residual chloroform content was ≤50 ppm. The flask was then placed in a vacuum drying oven (40°C, ≤10 mbar) for 2 h to completely remove residual solvent, yielding a lipid film loaded with the drug.

[0053] Step 2: Aqueous phase preparation

[0054] Take pH 6.5 phosphate buffer (PBS), add sophorobiose and ethoxyquinoline, stir to dissolve, and then sterilize through a 0.22 μm filter membrane to obtain an aqueous solution. The ratio of sophorobiose, ethoxyquinoline, and buffer solution is 10-18 g : 0.01-0.03 g : 300-500 mL (preferably 400 mL). Based on the lipid carrier feed mass (30-50 g), the ratio of aqueous phase volume to lipid mass is controlled at 6-16.7 mL / g, preferably 10 mL / g, to ensure that the lipid film mass to aqueous phase volume ratio is approximately 1:10, which is beneficial for forming uniform nanoliposomes. The overall material ratio is as follows: Lipid carrier: Eicosapentaenoic acid: Lipooxygenin B4: Phosphatidylserine: Dehydrorosilicate sophorbiose: Ethoxyquinoline: Buffer = 30-50g: 20-30g: 0.1-0.5g: 8-12g: 1-2g: 10-18g: 0.01-0.03g: 300-500mL. Preferably, the ratio is: Lipid carrier: Eicosapentaenoic acid: Lipooxygenin B4: Phosphatidylserine: Dehydrorosilicate sophorbiose: Ethoxyquinoline: Buffer = 40g: 25g: 0.3g: 10g: 1g: 15g: 0.02g: 400mL.

[0055] The aqueous solution was preheated to 55°C and slowly added to a flask containing a lipid membrane loaded with the drug. The mixture was stirred until the lipid membrane was hydrated.

[0056] Step 3: Ultrasonic dispersion to form nanoliposomes

[0057] The lipid film was fully hydrated to form a crude emulsion using a vortex mixer at low speed (200 rpm). The crude emulsion was then transferred to an ultrasonic cell disruptor, set to 200 W power and 40 kHz frequency, and ultrasonically treated for 15 minutes with a duty cycle of 3 seconds on, 3 seconds off, until the emulsion became translucent. The emulsion was then extruded and filtered three times using a liposome extruder (using a 100 nm pore size polycarbonate membrane) to obtain a uniform nanoliposome suspension with a particle size of 80-120 nm. The liposome extruder treatment breaks up the aggregation of the original liposomes, allowing liposomes of different sizes in the crude emulsion to pass through a polycarbonate membrane with a specific pore size, ultimately resulting in a homogeneous system with a narrow particle size distribution. The extrusion process reshapes the liposome bilayer structure, reduces membrane defects, and makes the liposomes more spherical and the membrane structure more stable.

[0058] Step 4: Freeze-drying and shaping

[0059] Add mannitol at a final concentration of 2-4% (preferably 3%, by mass) and trehalose at a final concentration of 1-3% (preferably 2%, by mass) to the nanoliposome suspension as freeze-drying protectants, mix well, and dispense into vials. Pre-freeze the vials by placing them in a -40°C freezer for 2 hours to form a stable solid state. Then perform vacuum freeze-drying, transferring the vials to a freeze dryer and drying at -40°C and a vacuum degree <10 Pa for 12 hours. Subsequently, increase the temperature to 25°C at a rate of 0.5°C / min, and continue drying for 2 hours after the temperature increase is complete. After completing the above steps, collect the finished product. The freeze-dried formulation is a loose powder, which is then sealed under nitrogen to prevent oxidation, thus obtaining the ω-3 fatty acid-lipoxygen-phospholipid complex regulatory formulation for intestinal inflammation in yellow catfish according to this scheme.

[0060] For preventative purposes, the compound regulatory agent prepared in Example 1 can be added to feed (e.g., Jiangtuan feed from Sichuan Zhonglian Chuang Biotechnology Co., Ltd.) at a final concentration of 0.1-0.2% (mass percentage). Feed twice daily for 5 consecutive days, or once monthly, suitable for the seedling stage and before and after transportation.

[0061] For therapeutic purposes, the compound regulatory agent prepared in Example 1 can be added to the feed at a final concentration of 0.3-0.4% (mass percentage). Administer three times daily for 10 consecutive days, in conjunction with water disinfection (such as potassium persulfate compound salt). This is suitable for the acute phase of inflammation.

[0062] Experimental Example

[0063] The intestinal inflammation-regulating preparations of yellow catfish containing ω-3 fatty acids, lipoxygen, and phospholipids were prepared according to the formulations in Tables 2 and 3 for each experimental and control group. The excipients and other solvents (soybean lecithin, cholesterol, chloroform-methanol mixed solvent, phosphate buffer, mannitol, and trehalose, etc.) were added according to the optimal method described in Example 1, and will not be repeated here.

[0064] Table 2: Formulation composition of active ingredients in the experimental and control groups

[0065]

[0066] Table 3: Formulation composition of active ingredients in the experimental and control groups

[0067]

[0068] The preparation methods for experimental groups 1-3 and control groups 1-9 are the same as the optimal method in Example 1. If a certain component is missing, simply omit the addition of that component in the corresponding step.

[0069] The formula for control group 10 is the same as that for experimental group 1, except for the preparation method, as detailed below:

[0070] Lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, dehydrorosinic acid, sophorobiose, and ethoxyquinoline were dissolved in a chloroform-methanol mixture and then rotary evaporated to obtain a drug-loaded lipid membrane. PBS at 55°C was then slowly added to the flask containing the drug-loaded lipid membrane, and the mixture was stirred until the lipid membrane was hydrated. The membrane was then ultrasonically dispersed and extruded to form nanoliposomes. The nanoliposomes were lyophilized to obtain a loose powdery compound preparation for regulating intestinal inflammation in yellow catfish containing ω-3 fatty acids, lipoxygenin, and phospholipids. Except for the operation methods described above, the remaining operation methods, technical parameters, and reagent dosages were exactly the same as the optimal process in Example 1. That is, in control group 10, all six active ingredients were added to the lipid carrier.

[0071] The formula for control group 11 is the same as that for experimental group 1, except for the preparation method, as detailed below:

[0072] Eicosapentaenoic acid, lipoxygenin B4, and phosphatidylserine were added to a lipid carrier to form a drug-loaded lipid membrane. Sophorabiose, ethoxyquinoline, and dehydrorosinic acid were added to PBS to form an aqueous solution. Except for the procedures described above, all other procedures, technical parameters, and reagent dosages were identical to the optimal process in Example 1. Specifically, in control group 11, dehydrorosinic acid was added to the aqueous solution instead.

[0073] The formula for control group 12 is the same as that for experimental group 1, except for the preparation method, as detailed below:

[0074] Eicosapentaenoic acid, lipoxygenase B4, and dehydrorosinic acid were added to a lipid carrier to form a drug-loaded lipid membrane. Sophorabiose, ethoxyquinoline, and phosphatidylserine were added to PBS to form an aqueous solution. Except for the procedures described above, all other procedures, technical parameters, and reagent dosages were identical to the optimal process in Example 1. Specifically, in control group 12, phosphatidylserine was added to the aqueous solution instead.

[0075] The formula for control group 13 is the same as that for experimental group 1, except for the preparation method, as detailed below:

[0076] Eicosapentaenoic acid, lipoxygenin B4, dehydrorosinic acid, phosphatidylserine, and ethoxyquinoline were added to the lipid carrier to form a drug-loaded lipid membrane. Sophora bisaccharide was added to PBS to form an aqueous solution. Except for the procedures described above, all other procedures, technical parameters, and reagent dosages were exactly the same as the optimal process in Example 1. That is, in control group 13, ethoxyquinoline was added to the lipid carrier instead.

[0077] The formulation of control group 14 is the same as that of experimental group 1, except for the preparation method, which is as follows: the aqueous solution is preheated to 50°C and slowly added to a flask containing a lipid membrane loaded with the drug, and mixed thoroughly to hydrate the lipid membrane. Except for the operation described above, the other operation methods, technical parameters, and reagent dosages are exactly the same as the optimal process in Example 1. That is, in control group 14, the preheating temperature of the aqueous solution is lowered.

[0078] The formulation of control group 15 was the same as that of experimental group 1, except for the preparation method, which is as follows: the aqueous solution was preheated to 60°C and slowly added to a flask containing a lipid membrane loaded with the drug, and mixed thoroughly to hydrate the lipid membrane. Except for the operation described above, all other operation methods, technical parameters, and reagent dosages were exactly the same as the optimal process in Example 1. That is, in control group 15, the preheating temperature of the aqueous solution was increased.

[0079] Taking experimental group 1 as an example, the obtained nanoliposome suspension was tested. The average size of the nanoparticles was around 100 nm, and the PDI (polydispersity index) was <0.2. The EPA encapsulation efficiency was 88.5%, and the lipoxygenin B4 encapsulation efficiency was 92.3%. Using lipid carriers to encapsulate EPA and lipoxygenin B4 facilitates the delivery of these substances and provides protection. The microbiological indicators of the lyophilized formulation were: total bacterial count <100 CFU / g, mold and yeast <10 CFU / g, and no pathogenic bacteria (Salmonella, Escherichia coli) were detected.

[0080] The intestinal inflammation-regulating compound preparations containing ω-3 fatty acids, lipoxygenin, and phospholipids, prepared from experimental groups 1-3 and control groups 1-15, were used to study the effects on Vibrio clearance, the promotion of ZO-1 protein expression in the intestinal mucosa, and the ROS clearance rate. The specific experimental methods are as follows:

[0081] For yellow catfish weighing 50±5g, artificial infection with Vibrio mimicry (1×10⁻⁶) was performed. 3 CFU / g body weight). After anesthetizing the fish, bacterial fluid was injected into the abdominal cavity (inner side of the pectoral fin base) using a sterile syringe for infection. Among the bacteria were *Vibrio mimicus* (…). Vibrio mimicATCC33653 is a commercially available model strain of Vibrio mimicry. The intestinal inflammation-regulating compound preparation containing ω-3 fatty acids, lipoxygen, and phospholipids, prepared for experimental groups 1-3 and control groups 1-15, was added to the feed of yellow catfish. The compound preparation was added at 0.4% (in conventional catfish feed). After the Vibrio mimicry infection model was successfully established, the fish were fed the medicated feed three times a day. The total daily feed intake, with or without the compound preparation, was 3% of the fish's body weight, with each feeding being 1% of the fish's body weight. After 5 days of continuous feeding (testing was conducted 5 days after infection), various indicators were measured. A control group (fish fed ordinary feed after infection) and a blank group (fish fed ordinary feed before infection) were also set up.

[0082] The specific testing indicators are as follows:

[0083] (1) Detection of intestinal villus height (H&E staining method)

[0084] Intestinal tissue from experimental animals was collected and quickly rinsed with physiological saline to avoid any residue. The tissue was cut into 0.5-1 cm segments and fixed in 4% paraformaldehyde fixative for at least 24 hours to ensure complete fixation. After dehydration, embedding, sectioning, and mounting, H&E staining was performed, followed by microscopic measurements. Using a microscope, at 400x magnification, 10 complete and clear intestinal villi from each sample were selected, and their height was measured using the microscope's built-in measurement software. The average value was taken as the intestinal villi height for that sample (single sample). Ten yellow catfish samples from each experimental group, control group, and blank group underwent the same test, and the average value was taken as the measurement value for that experimental group.

[0085] This indicator reflects the integrity of the intestinal mucosal structure and the state of nutrient absorption function. Intestinal villi are finger-like projections of the intestinal epithelium into the intestinal lumen; their height directly determines the absorptive surface area of ​​the intestinal mucosa, and the integrity of villi morphology is fundamental to the stability of the intestinal barrier structure. In the yellow catfish infection model, pathogenic bacteria damage intestinal epithelial cells, leading to villi atrophy, breakage, and shedding, resulting in reduced villi height, which in turn causes decreased nutrient absorption efficiency and increased intestinal barrier permeability. This indicator, by quantifying intestinal villi morphological parameters, objectively evaluates the protective or repairing effect of the compound regulatory agent on the intestinal mucosal structure. Its detection results can serve as morphological evidence of the intestinal mucosal protective effect of the compound regulatory agent, demonstrating its effectiveness in improving infection-induced intestinal structural damage.

[0086] (2) Detection of ZO-1 protein expression in intestinal mucosa (ELISA method)

[0087] Intestinal tissue was collected 5 days post-infection, and total protein was extracted. The protein concentration was determined using a BCA kit, and the total protein concentration was adjusted to 1 mg / mL. ZO-1 protein ELISA kit was used for detection (following the kit instructions). The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. A standard curve was plotted based on the OD values ​​of the standards, and the ZO-1 protein concentration in the samples was calculated. The relative expression values ​​of each experimental group, control group, and control group were calculated using the control group's histone expression level as a baseline (set to 1.00).

[0088] This indicator reflects the integrity of tight junctions in the intestinal epithelium (a core indicator of intestinal barrier function). ZO-1 (closed small ring protein 1) is a key structural protein of tight junctions between intestinal epithelial cells, and its expression level is directly related to the stability of tight junctions. As the core of the physical barrier of the intestinal barrier, tight junctions can prevent pathogens, toxins, and incompletely digested macromolecules from penetrating the epithelium and entering the circulatory system, thus maintaining intestinal homeostasis. Under infectious conditions, the virulence factors of pathogens degrade ZO-1 protein, leading to loosening of tight junctions, increased intestinal permeability, and thus exacerbating infection damage. This indicator quantifies the functional status of the intestinal barrier by detecting the relative expression level of ZO-1 protein. If the relative expression value of ZO-1 in the combined regulatory agent intervention group is significantly higher than that in the infected control group, it indicates that the combined regulatory agent can enhance the mechanical barrier function of the intestinal epithelium by upregulating the expression of tight junction proteins, reduce the transmembrane transport of pathogens and their metabolites, provide molecular-level evidence for the technical effect of the combined regulatory agent in improving intestinal barrier permeability, and provide supporting data for the combined regulatory agent in enhancing intestinal defense function.

[0089] (3) Number of Vibrio intestinal bacteria (plate count method)

[0090] Five days post-infection, 0.5g of intestinal tissue was collected and homogenized, followed by serial dilutions. 0.1mL of each dilution was spread onto TCBS agar plates and incubated at 28°C for 24 hours. Plates with colony counts between 30 and 300 were selected for counting, and the Vibrio load (CFU / g) per gram of intestinal tissue was calculated.

[0091] This indicator reflects the proliferation capacity of pathogenic bacteria in the intestine and the antibacterial / antimicrobial effect of the compound regulatory agent. Vibrio, as the pathogenic bacterium in the infection model, has a viral load (CFU / g) in the intestine that directly reflects the severity of the infection and the colonization and proliferation efficiency of the pathogenic bacteria. TCBS medium is a selective medium for Vibrio; through serial dilution plating and counting, the number of surviving Vibrio bacteria in intestinal tissue can be accurately quantified, serving as a functional indicator for evaluating whether the compound regulatory agent directly inhibits or indirectly suppresses the proliferation of pathogenic bacteria. The detection results of this indicator provide direct evidence of the effectiveness of the compound regulatory agent in combating intestinal pathogenic bacterial infections.

[0092] (4) Detection of interleukin-6 (IL-6) content in intestinal tissue (ELISA method)

[0093] Intestinal tissue (approximately 0.1 g) from infected yellow catfish was collected 5 days post-infection, rinsed thoroughly in pre-cooled physiological saline, blotted dry with filter paper, and weighed. The tissue was placed in a glass homogenizer, and pre-cooled PBS (pH 7.2-7.4) was added at a weight-to-volume ratio of 1:9 for mechanical homogenization in an ice bath. The homogenate was transferred to centrifuge tubes and centrifuged at 4°C and 5000 rpm for 10 min. The supernatant was carefully collected and stored at -80°C for later analysis. A fish-specific interleukin-6 (IL-6) ELISA kit (e.g., Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number: ML063133F) was used, strictly following the kit instructions. The simplified steps are as follows: Add the standard and tissue homogenate supernatant to the wells of the pre-coated antibody-impregnated ELISA plate, incubate, and wash; add the biotinylated detection antibody, incubate again, and wash; add horseradish peroxidase (HRP)-labeled streptavidin, incubate, and wash thoroughly; add the substrate TMB for colorimetric reaction, and finally add the stop solution to terminate the reaction. Immediately measure the absorbance (OD value) of each well at 450 nm using an ELISA reader. Plot a standard curve based on the standard concentration and corresponding OD value. Substitute the OD value of the sample into the standard curve to calculate the IL-6 content per unit weight of intestinal tissue (in pg / g). Simultaneously, determine the total protein concentration of each sample using the BCA method. The final result can also be corrected to pg / mg prot to eliminate errors caused by differences in protein concentration.

[0094] This indicator reflects the intensity and degree of local inflammatory response in the intestine. IL-6 is a typical pro-inflammatory cytokine. After pathogenic bacterial infection, intestinal mucosal immune cells (such as macrophages and lymphocytes) are activated and secrete large amounts of IL-6. The change in its content is positively correlated with the severity of intestinal inflammatory response. Overexpression of IL-6 can exacerbate inflammatory damage to intestinal epithelial cells and disrupt intestinal mucosal homeostasis; while proper regulation of the inflammatory response is key to reducing tissue damage caused by infection.

[0095] This indicator assesses the anti-inflammatory activity of the compound regulatory agent by quantifying the absolute content of IL-6 in intestinal tissue. If the IL-6 content in the compound regulatory agent intervention group is significantly lower than that in the infection control group, it indicates that the compound regulatory agent can regulate the intensity of local intestinal inflammation by inhibiting the secretion of pro-inflammatory cytokines, thereby reducing damage to the intestinal mucosa by inflammatory factors and achieving an anti-inflammatory and intestinal-protective effect. The detection results provide immunomolecular evidence for the technical efficacy of the compound regulatory agent in improving infection-induced intestinal inflammation.

[0096] (5) Detection of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in intestinal tissue

[0097] Intestinal tissue (approximately 0.1g) from the same location was collected from yellow catfish 5 days post-infection. The tissue was rinsed thoroughly in pre-cooled physiological saline, blotted dry with filter paper, and weighed. The tissue was placed in a glass homogenizer, and pre-cooled physiological saline was added at a weight-to-volume ratio of 1:9 for mechanical homogenization in an ice bath to prepare a 10% tissue homogenate. The homogenate was centrifuged at 4°C and 2500-3000 rpm for 10-15 minutes, and the supernatant was carefully collected for later use.

[0098] SOD activity assay: The Xanthine oxidase method (hydroxylamine method) SOD assay kit was used. The main principle is that SOD inhibits superoxide anion free radicals (O2·) - The oxidation of hydroxylamine was assessed, and SOD activity was calculated by measuring the absorbance of the reaction system. The procedure was strictly followed according to the kit instructions, using a spectrophotometer to measure the absorbance (OD value) at 550 nm. SOD activity per unit weight of intestinal tissue was calculated using the formula, expressed as U / mg prot.

[0099] MDA content determination: The procedure was performed using a thiobarbituric acid (TBA) method MDA assay kit. The main principle is that MDA condenses with TBA under acidic conditions to form a red product with a maximum absorption peak at 532 nm. The procedure was strictly followed according to the kit instructions, and the absorbance (OD value) was measured at 532 nm using a spectrophotometer. The MDA content per unit weight of intestinal tissue was calculated using the formula, expressed as nmol / mg prot.

[0100] This indicator reflects the oxidative stress state and antioxidant capacity of intestinal tissue. Superoxide dismutase (SOD) is an important endogenous antioxidant enzyme in the body. Its core function is to catalyze the dismutation of superoxide anion free radicals into hydrogen peroxide, which is then cleared by other antioxidant systems, reducing the oxidative damage of free radicals to cellular lipids, proteins, and nucleic acids. It is a key enzyme in maintaining the body's oxidation-antioxidant balance. In the infection model, pathogenic bacterial invasion triggers oxidative stress in intestinal tissue, leading to a large accumulation of free radicals, and SOD activity decreases due to depletion. The activity level of this indicator directly reflects the antioxidant capacity of intestinal tissue: the SOD activity in the drug intervention group was significantly higher than that in the infection control group, indicating that the drug can enhance the activity of endogenous antioxidant enzymes, improve the ability of intestinal tissue to clear free radicals, and reduce the damage of oxidative stress to intestinal epithelial cells. MDA is the end product of lipid peroxidation, and its content is positively correlated with the degree of cellular oxidative damage: when free radicals attack unsaturated fatty acids on the cell membrane, a lipid peroxidation chain reaction is triggered, leading to cell membrane structure destruction, and the large-scale generation and accumulation of MDA. The level of this indicator directly reflects the degree of oxidative damage to intestinal tissue: the MDA content in the drug intervention group was significantly lower than that in the infection control group, indicating that the drug can inhibit the occurrence of lipid peroxidation, reduce the accumulation of oxidative damage products, and protect the integrity of intestinal cell membranes.

[0101] SOD activity and MDA content are complementary verification indicators of oxidative stress-antioxidant damage: joint detection of the two can comprehensively reflect the antioxidant effect of the drug.

[0102] In summary, the above indicators, based on five dimensions—structural morphology (intestinal villus height), barrier function (ZO-1), pathogen clearance (Vibrio load), inflammation regulation (IL-6), and oxidative damage protection (SOD / MDA)—constructed a multi-target evaluation system for the effects of drugs on infectious intestinal injury. Detailed experimental results are shown in Tables 4 and 5.

[0103] Table 4: Results of various indicators 5 days after infection (blank group, experimental group, control group 1-6; mean ± standard deviation, n=10)

[0104]

[0105] Table 5: Results of various indicators 5 days after infection (control group, comparison group 7-15; mean ± standard deviation, n=10)

[0106]

[0107] (1) Overall effect of the compound regulatory preparation for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids in this scheme

[0108] Experimental groups 1-3 represent the implementation methods of this scheme. The dosage of their active ingredients is within the optimal range, and all test indicators show ideal results, which are significantly better than the subsequent comparative groups and the control group, fully verifying the scientific nature and feasibility of the formulation system of this scheme.

[0109] Compared with the blank group and the control group, the compound regulatory agents prepared in experimental groups 1-3 have the following effects:

[0110] Intestinal structure protection: The heights of intestinal villi in experimental groups 1-3 were 681.5±11.7μm, 654.8±14.9μm, and 667.6±13.8μm, respectively. Although these were lower than those in the uninfected control group (753.8±8.2μm), they were much higher than those in the infected control group (476.8±11.3μm) and significantly better than most control groups. This indicates that the preparation can effectively reduce intestinal villi atrophy and breakage caused by Vibrio infection and maintain the integrity of the intestinal mucosal structure.

[0111] Enhanced barrier function: The relative expression levels of ZO-1 protein were 1.823±0.148, 1.701±0.121, and 1.752±0.139, respectively, all of which were significantly higher than those in the control group, demonstrating that the formulation can effectively upregulate the expression of intestinal tight junction proteins, strengthen the intestinal mechanical barrier function, and reduce the transmembrane transport of pathogens.

[0112] Significant antibacterial effect: the intestinal Vibrio load was 1.52±0.31×10⁻⁶. 5 CFU / g, 2.08±0.39×10 5 CFU / g, 1.79±0.32×10 5 CFU / g, compared with the control group (4.69±0.51×10⁻⁶). 5 The CFU / g decreased by 54.8%-67.6%, indicating that the preparation can effectively inhibit the colonization and proliferation of Vibrio in the intestine.

[0113] The anti-inflammatory effect was outstanding: the intestinal IL-6 levels were 38.45±4.15 pg / mg prot, 45.18±5.03 pg / mg prot, and 41.76±4.42 pg / mg prot, respectively, which were significantly lower than those in the control group (95.75±10.15 pg / mg prot) and close to the level of the blank group (25.10±2.75 pg / mg prot). This indicates that the preparation can effectively regulate the local inflammatory response in the intestine and reduce the damage of inflammatory factors to the mucosa.

[0114] Excellent antioxidant capacity: The SOD activity (78.6-85.2 U / mg prot) of experimental groups 1-3 was significantly higher than that of the control group (50.7±9.8 U / mg prot), and the MDA content (1.21-1.79 nmol / mg prot) was significantly lower than that of the control group (4.02±0.68 nmol / mg prot), indicating that the preparation can reduce the intestinal oxidative stress damage caused by infection by regulating the oxidation-antioxidant balance.

[0115] It is evident that the dosage range of the formulas in experimental groups 1-3 (EPA 20-30 parts, lipoxygenase B4 0.1-0.5 parts, PS 8-12 parts, dehydrorosinic acid 1-2 parts, sophorobiose 10-18 parts, ethoxyquinoline 0.01-0.03 parts) is scientifically reasonable and exhibits excellent synergistic effects in terms of intestinal structure protection, barrier function enhancement, antibacterial, anti-inflammatory, and antioxidant properties.

[0116] (2) Synergistic effect analysis of EPA, lipoxygenase B4 and PS

[0117] The necessity of using the three together

[0118] Control groups 1-3 lacked EPA, PS, and lipoxygenase B4, respectively, while the remaining components and preparation processes were the same as experimental group 1. The differences in their performance indicators compared to experimental group 1 directly reflect the impact of the absence of a single component on the overall effect. Compared to experimental group 1, the performance indicators of all three groups showed significant deterioration after their absence. All indicators in control groups 1-3 were significantly worse than those in experimental group 1. The absence of any one component led to a substantial decrease in the function of the formulation, with the absence of EPA having the most significant impact. This demonstrates that EPA, lipoxygenase B4, and PS are functionally interdependent and indispensable, forming a key combination for the formulation to exert its core effect.

[0119] Strengthening verification of the synergistic effect of the three

[0120] Control groups 4-6 were each missing two components, while the dosages of the remaining components were the same as in experimental group 1: control group 4 used EPA alone, control group 5 used PS alone, and control group 6 used lipoxygenase B4 alone. The results showed that the deterioration of the indicators was more significant after the absence of two components than after the absence of a single component.

[0121] The synergistic effects of the three compounds on six technical indicators were calculated and expressed using synergy coefficients. Synergy coefficient = Measured combined effect value / Theoretical combined effect value; synergy coefficient > 1 indicates synergistic effect; synergy coefficient = 1 indicates additive effect; synergy coefficient < 1 indicates antagonistic effect. According to the Bliss model, the theoretical combined effect value of the three compounds is: Etheoretical = E(A) + E(B) + E(C) - E(A)E(B) - E(A)E(C) - E(B)E(C) + E(A)E(B)E(C); where A represents EPA, B represents PS, and C represents lipoxygenin B4. For intestinal villus height, relative ZO-1 expression, and SOD activity (higher values ​​indicate better effects), E(A) or E(B) or E(C) or Emeasured (measured combined effect value) is calculated as: (Sample value of control group 4 or 5 or 6 or experimental group 1 - control group value) / (blank group value - control group value) × 100%. For the indicators of Vibrio ingrainiensis count, IL-6 content, and MDA content (lower values ​​indicate better efficacy), the calculation method for E(A), E(B), E(C), or E (measured combined effect value) is: (control group value - sample value of control group 4, 5, or 6, or experimental group 1) / (control group value - blank group value) × 100%. Synergy coefficients were calculated for the above six indicators, and the results were all > 1. Among them, the synergy coefficients for Vibrio ingrainiensis count, relative ZO-1 expression level, and IL-6 content were more significant (approximately 1.7, 1.5, and 1.2, respectively).

[0122] This demonstrates that EPA, PS, and lipoxygenin B4, in combination, upregulate tight junction protein expression, strengthening the intestinal mechanical barrier; compensate for the insufficient antibacterial effect of EPA alone, inhibiting Vibrio colonization and proliferation through synergistic action; synergistically inhibit the secretion of pro-inflammatory factors, regulating the intensity of local intestinal inflammatory responses; synergistically inhibit lipid peroxidation, reducing the accumulation of oxidative damage products; jointly maintain the integrity of the intestinal mucosal structure, alleviating villous atrophy caused by Vibrio infection; and synergistically enhance antioxidant enzyme activity, assisting in the scavenging of free radicals in the intestine. The three components exhibit synergistic effects on five main targets: strengthening intestinal barrier function (ZO-1), inhibiting pathogenic bacteria (Vibrio count), regulating inflammation (IL-6), protecting against oxidative damage (MDA / SOD), and protecting mucosal structure (villous height), forming a multi-target synergistic regulatory system. The effects of single components or binary combinations are significantly inferior to those of the three-component combination (synergistic coefficients > 1), proving that the combined technical solution is not a simple additive approach, but rather achieves amplified effects through synergistic action, yielding unexpected technical results.

[0123] (3) Analysis of the combined effects of sophorobiose and ethoxyquinoline

[0124] Sophora bisaccharide and ethoxyquinoline are existing antioxidants. Their combined use in this treatment environment produced a multifaceted synergistic effect, as detailed in the experimental data of control groups 7 and 8. According to the Bliss model, the theoretical combined effect of the two factors is: Etheoretical = E(A) + E(B) - E(A)E(B); where A represents sophora bisaccharide and B represents ethoxyquinoline. Synergistic coefficients were calculated for the six indicators, and all results were >1. The synergistic coefficients for the intestinal Vibrio count and IL-6 content were particularly significant (around 1.1). Sophora bisaccharide and ethoxyquinoline exhibited synergistic effects across all six indicators, with significant synergistic effects in inflammation regulation (IL-6), forming a multi-dimensional synergistic system of antioxidation, anti-inflammation, barrier protection, and antibacterial activity. Based on their complementary properties, they act on different targets such as oxidative stress, inflammatory response, and intestinal structure. Their synergistic effect is not a simple additive effect of a single component, but rather an amplification of effects through functional complementarity, resulting in unexpected technical benefits.

[0125] (4) Analysis of the effects of dehydrorosin acid

[0126] Control group 9 lacks dehydrorosin acid, while the dosage and preparation process of the other components are the same as those in experimental group 1. The difference in its performance indicators compared to experimental group 1 directly reflects the function of dehydrorosin acid.

[0127] Regarding intestinal villus height, the height of control group 9 was 557.2±19.8μm, a decrease of 18.2% compared to 681.5±11.7μm in experimental group 1, indicating more severe intestinal mucosal damage; the relative expression level of ZO-1 was 1.198±0.097, a decrease of 34.3% compared to experimental group 1, indicating increased intestinal barrier permeability; and the intestinal Vibrio load was 4.03±0.49×10⁻⁶. 5 The CFU / g level was 165.1% higher than that in experimental group 1, indicating a significant increase in the proliferation capacity of pathogenic bacteria; the IL-6 level was 75.14±8.05 pg / mg prot, 95.4% higher than that in experimental group 1, indicating an aggravated inflammatory response; the SOD activity was 62.8±7.8 U / mg prot, 26.3% lower than that in experimental group 1, indicating a decrease in the consumption of antioxidant enzyme activity; and the MDA level was 2.73±0.49 nmol / mg prot, 125.6% higher than that in experimental group 1, indicating the accumulation of lipid peroxidation products and aggravated cell damage.

[0128] The core function of dehydrorosinic acid is to directly inhibit the proliferation of Vibrio in the intestine. In this regimen, in addition to its function as an antibacterial agent, it also enhances the intestinal barrier function (ZO-1), inflammation regulation (IL-6), oxidative damage protection (MDA / SOD), and mucosal structure protection (villi height).

[0129] (5) The effect of preparation process on the efficacy of the formulation

[0130] The formulations of control groups 10-15 were completely identical to those of experimental group 1, with the only difference being a single preparation process parameter. The specific differences and performance indicators are as follows:

[0131] The key role of component distribution method (comparison groups 10-13)

[0132] The optimized component allocation scheme for experimental group 1 was as follows: EPA, lipoxygenase B4, PS, and dehydrorosinic acid were added to the lipid phase, while sophorobiose and ethoxyquinoline were added to the aqueous phase. The component allocation schemes for control groups 10-13 differed.

[0133] In control group 10, all components (including sophorobiose and ethoxyquinoline) were added to the lipid phase, and the remaining process parameters were the same as in experimental group 1. The results showed that control group 10 performed the worst in all aspects: intestinal villus height was 519.4±24.6μm, a decrease of 23.8% compared to experimental group 1; relative ZO-1 expression was 1.047±0.079, a decrease of 42.6%; and intestinal Vibrio load was 4.79±0.58×10⁻⁶. 5 CFU / g increased by 215.1%; IL-6 content was 82.36±8.83 pg / mg prot, increased by 114.2%; SOD activity was 58.5±8.7 U / mg prot, decreased by 31.3%; MDA content was 3.02±0.57 nmol / mg prot, increased by 149.6%.

[0134] Control group 11 had dehydrorosin acid added to the aqueous phase instead of the control group 1; the remaining processes were the same as in experimental group 1. The results showed: intestinal villus height 634.5±14.9 μm, a decrease of 6.9% compared to experimental group 1; relative ZO-1 expression level 1.551±0.128, a decrease of 14.9%; and intestinal Vibrio load 1.98±0.29×10⁻⁶. 5 CFU / g increased by 30.3%; IL-6 content increased by 52.4% to 58.61±6.24 pg / mg prot; SOD activity decreased by 13.3% to 73.9±5.9 U / mg prot; MDA content increased by 39.7% to 1.69±0.35 nmol / mg prot.

[0135] Control group 12 had PS replaced with an aqueous phase, while the rest of the process was the same as experimental group 1. The results showed: intestinal villus height 544.3±21.7μm, a decrease of 20.1% compared to experimental group 1; relative ZO-1 expression level 1.146±0.091, a decrease of 37.1%; and intestinal Vibrio load 3.76±0.48×10⁻⁶. 5CFU / g increased by 147.4%; IL-6 content increased by 91.9% to 73.79±7.85 pg / mg prot; SOD activity decreased by 24.6% to 64.2±7.7 U / mg prot; MDA content increased by 99.2% to 2.41±0.48 nmol / mg prot.

[0136] In control group 13, ethoxyquinoline was replaced with a lipid phase, while the rest of the process was the same as in experimental group 1. The results showed: intestinal villus height was 614.6±16.8 μm, a decrease of 9.8% compared to experimental group 1; relative ZO-1 expression was 1.576±0.121, a decrease of 13.5%; and intestinal Vibrio load was 1.91±0.28×10⁻⁶. 5 CFU / g increased by 25.7%; IL-6 content increased by 36.2% to 52.37±5.58 pg / mgprot; SOD activity decreased by 10.1% to 76.6±5.2 U / mgprot; and MDA content increased by 33.9% to 1.62±0.29 nmol / mgprot.

[0137] Optimized range of hydration temperature (comparison group 14-15)

[0138] The preheating temperature of the aqueous phase in experimental group 1 was 55℃, while the preheating temperature of the aqueous phase in control group 14 was lowered to 50℃, and that in control group 15 was raised to 60℃. All other process parameters were the same as those in experimental group 1.

[0139] The indicators of control group 14 (50℃) were as follows: intestinal villus height 647.7±13.6μm, a decrease of 4.9% compared with experimental group 1; relative expression of ZO-1 1.781±0.137, a decrease of 2.3%; and intestinal Vibrio load 1.68±0.27×10⁻⁶. 5 CFU / g increased by 10.5%; IL-6 content increased by 7.0% to 41.15±4.38 pg / mg prot; SOD activity decreased by 1.6% to 83.8±4.9 U / mg prot; and MDA content increased by 14.0% to 1.38±0.28 nmol / mg prot. 50℃ still met the lipid membrane hydration requirements, only slightly affecting the uniformity of liposome formation, with minimal impact on the overall effect, which is within an acceptable range.

[0140] The indicators of control group 15 (60℃) were as follows: intestinal villus height 644.8±14.7μm, a decrease of 5.4% compared with experimental group 1; relative expression of ZO-1 1.749±0.129, a decrease of 4.1%; and intestinal Vibrio load 1.81±0.29×10⁻⁶. 5CFU / g increased by 19.1%; IL-6 content increased by 13.8% to 43.76±4.65 pg / mg prot; SOD activity decreased by 4.3% to 81.5±5.1 U / mg prot; and MDA content increased by 24.8% to 1.51±0.30 nmol / mg prot. 60℃ did not cause lipid membrane degradation; only a small amount of components showed decreased stability due to excessively high temperature. The effect was slightly lower than that of experimental group 1, but still significantly better than other groups with abnormal processes.

[0141] In summary, EPA, lipoxygenase B4, PS, and dehydrorosinic acid must be added to the lipid phase, while sophorobiose and ethoxyquinoline must be added to the aqueous phase. This partitioning method improves component stability and bioavailability, which is a prerequisite for the formulation to exert its effect. The preheating temperature of the aqueous phase should be controlled at 50-60℃, preferably 55℃. This temperature range ensures sufficient hydration of the lipid membrane, forming uniform (80-120nm) and structurally stable nanoliposomes, avoiding insufficient hydration due to excessively low temperatures or component degradation due to excessively high temperatures.

[0142] (6) Summary

[0143] This experiment, through a multi-group control design, comprehensively verified the technical advantages of compound regulatory formulations from three dimensions: synergistic effect of formulation, function of key components, and preparation process.

[0144] Synergistic effects of the formula: EPA, lipoxygenase B4, and PS are all indispensable and work together to regulate intestinal barrier function and inflammatory response; sophorobiose and ethoxyquinoline reduce oxidative stress damage through antioxidant synergistic effects; dehydrorosinic acid, as the core antibacterial component, forms a synergistic system of antibacterial, defense, and repair with the above components.

[0145] Key ingredient functions: Dehydrorosinic acid is the core of direct antibacterial action, EPA is the core of synergistic regulation, PS is the core of barrier function enhancement, lipoxygenase B4 is the core of anti-inflammatory action, and sophorobiose and ethoxyquinoline are the core of antioxidant action. Each ingredient performs its own function and works together with others.

[0146] Key to process optimization: Precise lipid / aqueous phase distribution of the ingredients and hydration temperature of around 55°C are the core process parameters to ensure the efficacy of the formulation, directly affecting the encapsulation efficiency, stability and bioavailability of the ingredients.

[0147] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A compound regulatory agent for intestinal inflammation in yellow catfish containing ω-3 fatty acids, lipoxygenin, and phospholipids, characterized in that: By weight, its active ingredients are: 20-30 parts eicosapentaenoic acid, 0.1-0.5 parts lipoxygenase B4, 8-12 parts phosphatidylserine, 1-2 parts dehydrorosinic acid, 10-18 parts sophorobiose, and 0.01-0.03 parts ethoxyquinoline.

2. The compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygenase, and phospholipids according to claim 1, characterized in that: The phosphatidylserine is of the 14:0 / 14:0 type.

3. The compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygenase, and phospholipids according to claim 1, characterized in that: By weight, its active ingredients are: 25 parts eicosapentaenoic acid, 0.3 parts lipoxygenase B4, 10 parts phosphatidylserine, 1 part dehydrorosinic acid, 15 parts sophorobiose, and 0.02 parts ethoxyquinoline.

4. A method for preparing a compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygenase, and phospholipids according to any one of claims 1-3, characterized in that: Includes the following steps: S1: Lipid membrane preparation The lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, and dehydrorosinic acid were added to a chloroform-methanol mixed solvent to obtain a lipid solution; the lipid solution was subjected to rotary evaporation to obtain a lipid membrane loaded with the drug. The lipid carrier is composed of soybean lecithin and cholesterol in a mass ratio of 60-80:20-40; S2: Aqueous phase preparation Sophora bisaccharide and ethoxyquinoline were added to a buffer solution and mixed to obtain an aqueous solution. The aqueous solution was preheated and then added to a container containing a lipid membrane loaded with the drug. The mixture was stirred until the lipid membrane was hydrated. S3: Preparation of nanoliposome suspension The hydrated lipid membrane forms a crude emulsion, which is then subjected to ultrasonic treatment and liposome extrusion to obtain a nanoliposome suspension. S4: Freeze-drying molding Mannitol and trehalose were added to the nanoliposome suspension, and after vacuum freeze-drying, a compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids, lipoxygen, and phospholipids was obtained.

5. The preparation method of the compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids according to claim 4, characterized in that: In S1, the mass ratio of lipid carrier, eicosapentaenoic acid, lipoxygenin B4, phosphatidylserine, and dehydrorosinic acid is 30-50: 20-30: 0.1-0.5: 8-12: 1-2.

6. The preparation method of the compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids according to claim 5, characterized in that: In S1, the ratio of lipid carrier to chloroform-methanol mixed solvent is 40-60g: 200-400mL; the chloroform-methanol mixed solvent is formed by mixing chloroform and methanol in a volume ratio of 1-2: 1-2. The rotary evaporation process is as follows: under reduced pressure at 60°C, the solvent is removed by rotary evaporation until a uniform lipid film is formed on the inner wall of the flask; nitrogen is purged for 30 min at 50°C and 0.2 MPa pressure, ensuring that the residual chloroform content is ≤50 ppm; then, the film is vacuum dried at 40°C for 2 h to remove the residual solvent and obtain the lipid film loaded with the drug.

7. The preparation method of the compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids according to claim 4, characterized in that: In S2, the ratio of sophorobiose, ethoxyquinoline, and buffer solution is 10-18 g : 0.01-0.03 g : 300-500 mL; After the aqueous solution is preheated to 55°C, lipid membrane hydration is performed. The buffer solution is a phosphate buffer.

8. The preparation method of the compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids according to claim 4, characterized in that: In S3, the ultrasonic processing parameters are 200W power and 40kHz frequency, and the ultrasonic processing is performed for 15 minutes according to the program of 3 seconds of operation and 3 seconds of rest. The liposome extrusion process used a polycarbonate membrane with a pore size of 100 nm and was filtered three times.

9. The preparation method of the compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids according to claim 4, characterized in that: In S4, mannitol at a final concentration of 2-4% and trehalose at a final concentration of 1-3% were added to the nanoliposome suspension as freeze-drying protectants. The vacuum freeze-drying procedure is as follows: pre-freeze at -40℃ for 2 hours; then dry at -40℃ and vacuum degree <10Pa for 12 hours, followed by heating to 25℃ at a rate of 0.5℃ / min; after the heating is completed, continue drying for 2 hours.

10. The use of the compound regulatory preparation of yellow catfish containing ω-3 fatty acid-lipoxygen-phospholipid as described in any one of claims 1-3 in the preparation of a drug for relieving intestinal inflammation in yellow catfish caused by Vibrio mimicry infection.

11. The use of the yellow catfish intestinal inflammation complex regulatory preparation containing ω-3 fatty acids-lipoxygen-phospholipids as described in any one of claims 1-3 in the preparation of a drug for inhibiting Vibrio mimicry infection.

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