Preparation method of compound pelteobagrus fulvidraco intestinal inflammation regulating preparation containing omega-3 fatty acid-lipoxin-phospholipid

Abstract

The invention relates to the technical field of prevention and control of aquatic diseases, in particular to a preparation method of a pelteobagrus fulvidraco intestinal inflammation composite regulation preparation containing omega-3 fatty acid-lipoxin-phospholipid. The composite regulation and control preparation is prepared from the following functional components: 20 to 30 parts of eicosapentaenoic acid, 0.1 to 0.5 part of lipoxin B4, 8 to 12 parts of phosphatidylserine, 1 to 2 parts of dehydroabietic acid, 10 to 18 parts of sophorobiose and 0.01 to 0.03 part of ethoxyquin. The product integrates the functions of resisting bacteria, promoting inflammation regression and promoting tissue repair through a multi-target synergistic mechanism to construct a complete inflammation regulation and control pathway; meanwhile, by virtue of a nano-liposome dosage form technology, the active ingredients are effectively protected; the product is completely compounded from natural components to realize an antibacterial effect. According to the technical scheme, the technical problems of low antibacterial efficiency, single action mechanism, insufficient mucous membrane repair capability and the like of the existing prevention and control technology for the intestinal inflammation of the pelteobagrus fulvidraco can be solved, and the pelteobagrus fulvidraco 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: (1) Natural antibacterial ingredients are inefficient: Although single plant essential oils (such as thymol, cinnamaldehyde, etc.) commonly used in the prior art have certain natural antibacterial activity, they generally have the problem of narrow antibacterial spectrum. For example, the inhibitory effect on Vibrio, a pathogenic bacterium that causes high incidence of intestinal inflammation in yellow catfish, the minimum inhibitory concentration (MIC) of such single component is usually ≥100 μg / mL, which is much higher than the effective concentration range that is easy to add in actual aquaculture. It cannot quickly control the spread of pathogens in the early stage of inflammation outbreak, and it is difficult to contain the spread of the disease.

[0006] (2) The inflammation-oxidative stress linkage is not blocked: After the intestinal infection of yellow catfish occurs, the pathogen stimulation will trigger the explosive production of reactive oxygen species (ROS) in intestinal epithelial cells. Excessive ROS not only directly damages intestinal mucosal cells, but also activates inflammatory signaling pathways, promoting the secretion of interleukin-6 (IL-6) like factors, tumor necrosis factor and other inflammatory factors. The release of inflammatory factors further exacerbates the generation of ROS, forming an oxidative stress-inflammation positive feedback loop, accelerating intestinal damage. Most existing control agents only regulate a single link in the inflammation pathway or oxidative stress pathway, and fail to fundamentally block the linkage mechanism of the two, especially the inhibition of key pro-inflammatory factors such as interleukin-6 (IL-6) is limited, resulting in unsatisfactory inflammation control effect.

[0007] (3) Insufficient intestinal mucosa 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 that maintain the tight junction between intestinal epithelial cells and ensure the function of the mucosal barrier. Most existing non-antibiotic preparations focus on inhibiting bacteria or reducing inflammation, and lack active ingredients that can directly promote the synthesis of tight junction proteins by yellow catfish intestinal epithelial cells, resulting in slow intestinal mucosa repair. Even if the short-term inflammation is relieved, chronic inflammation is likely to recur due to weak mucosal barrier function, and long-term control of intestinal health cannot be achieved.

[0008] In summary, the existing prevention and control technology for intestinal inflammation in yellow catfish has the problems of low antibacterial efficiency, single action mechanism, and insufficient mucosa repair capacity. Therefore, it is urgent to develop a complex control agent that can simultaneously achieve high-efficiency antibacterial, block oxidative stress-inflammation linkage, and promote intestinal mucosa repair, in order to meet the healthy development needs of yellow catfish aquaculture industry. SUMMARY

[0009] The present application aims to provide a complex control agent for intestinal inflammation in yellow catfish containing omega-3 fatty acids-lipoxins-phospholipids, to solve the technical problems of low antibacterial efficiency, single action mechanism, and insufficient mucosa repair capacity of the existing prevention and control technology for intestinal inflammation in yellow catfish.

[0010] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows: The compound control agent for intestinal inflammation of Pelteobagrus vachelli contains omega-3 fatty acid-lipoxin-phospholipid, and the effective components include 20-30 parts of eicosapentaenoic acid, 0.1-0.5 parts of lipoxin B4, 8-12 parts of phosphatidylserine, 1-2 parts of dehydroabietic acid, 10-18 parts of sophorobiose and 0.01-0.03 parts of ethoxyquin, by weight.

[0011] In summary, the technical principle of the present application is as follows: Further, the phosphatidylserine is phosphatidylserine of 14:0 / 14:0 type.

[0012] Further, the compound control agent for intestinal inflammation of Pelteobagrus vachelli contains omega-3 fatty acid-lipoxin-phospholipid, and the effective components include 25 parts of eicosapentaenoic acid, 0.3 parts of lipoxin B4, 10 parts of phosphatidylserine, 1 part of dehydroabietic acid, 15 parts of sophorobiose and 0.02 parts of ethoxyquin, by weight.

[0013] The present application also provides a preparation method of the compound control agent for intestinal inflammation of Pelteobagrus vachelli containing omega-3 fatty acid-lipoxin-phospholipid, comprising the following steps: S1: Lipid film preparation The lipid carrier, eicosapentaenoic acid, lipoxin B4, phosphatidylserine and dehydroabietic acid are added into a chloroform-methanol mixed solvent for mixing to obtain a lipid solution; the lipid solution is subjected to rotary evaporation treatment to obtain a drug-loaded lipid film; S2: Water phase preparation The sophorobiose and ethoxyquin are added into a buffer solution for mixing to obtain a water phase solution; after preheating, the water phase solution is added into a container containing the drug-loaded lipid film, and is uniformly mixed to hydrate the lipid film; S3: Preparation of nanoliposome suspension The hydrated lipid film forms a coarse emulsion, and the coarse emulsion is subjected to ultrasonic treatment and liposome extrusion to obtain a nanoliposome suspension; S4: Freeze-drying molding The nanoliposome suspension is added with mannitol and trehalose, and after vacuum freeze-drying, the compound control agent for intestinal inflammation of Pelteobagrus vachelli containing omega-3 fatty acid-lipoxin-phospholipid is obtained.

[0014] Further, in S1, the mass ratio of the lipid carrier, eicosapentaenoic acid, lipoxin B4, phosphatidylserine and dehydroabietic acid is 30-50:20-30:0.1-0.5:8-12:1-2.

[0015] Further, in S1, the lipid carrier is composed of soybean phospholipid and cholesterol with a mass ratio of 60-80:20-40; The ratio of the amount of the lipid carrier to the amount of the chloroform-methanol mixed solvent is 40-60 g:200-400 mL; the chloroform-methanol mixed solvent is formed by mixing chloroform and methanol at a volume ratio of 1-2:1-2; The method of the rotary evaporation treatment is: under the condition of 60 DEG C and reduced pressure, the solvent is removed by rotary evaporation until a uniform lipid film is formed on the inner wall of the flask; under the condition of 50 DEG C and 0.2 MPa pressure, nitrogen is blown for 30 min, and the residual amount of chloroform is ensured to be less than or equal to 50 ppm; then, under the condition of 40 DEG C, vacuum drying is performed for 2 h to remove the residual solvent, and a drug-loaded lipid film is obtained.

[0016] Further, in S2, the ratio of the amount of sophorose, ethoxyquinoline and buffer is 10-18 g:0.01-0.03 g:300-500 mL; After the aqueous solution is preheated to 55 DEG C, the lipid film hydration operation is performed The buffer is a phosphate buffer.

[0017] Further, in S3, the ultrasonic treatment parameters are power 200 W, frequency 40 kHz, and the ultrasonic treatment is performed for 15 min according to the program of working for 3 seconds and intermittently for 3 seconds; The liposome extrusion operation adopts a polycarbonate membrane with a pore size of 100 nm, and is extruded and filtered for 3 times.

[0018] Further, in S4, 2-4% of mannitol and 1-3% of trehalose are added to the nanoliposome suspension as freeze-drying protectants; The program of the vacuum freeze-drying is: pre-freezing for 2 h at-40 DEG C; then, drying for 12 h under the condition of-40 DEG C and vacuum degree less than 10 Pa, and then increasing the temperature to 25 DEG C at a rate of 0.5 DEG C / min; after the temperature increasing is completed, drying is continuously performed for 2 h.

[0019] The technical scheme also provides application of the complex regulator containing omega-3 fatty acid-lipoxin-phospholipid in preparation of a medicament for relieving intestinal inflammation of yellow-head catfish caused by Vibrio mimicus infection, or in preparation of a medicament for inhibiting Vibrio mimicus infection.

[0020] The technical principle of the technical scheme is that: The present application realizes multi-target point regulation of intestinal inflammation of yellow-head catfish by constructing a ternary synergistic system of omega-3 fatty acid, lipoxin and phospholipid, and combining with nanoliposome delivery technology.

[0021] Dehydroabietic acid in the plant antibacterial complex can destroy the lipid bilayer of the bacterial cell membrane, and its minimum inhibitory concentration (MIC) for Vibrio mimicus is 15 μg / mL, directly inhibiting the proliferation of pathogenic bacteria; eicosapentaenoic acid (EPA) in ω-3 fatty acids can reduce the secretion of hemolysin and other virulence factors by inhibiting the quorum sensing system of Vibrio, thereby reducing intestinal damage from two paths of killing pathogenic bacteria and blocking virulence. Lipoxin B4, a pro-resolution mediator of inflammation, can trigger the resolution of inflammation by specifically activating the receptor of the inflammation pathway, promote macrophages to clear apoptotic cells, and down-regulate the pro-inflammatory response to achieve precise termination of inflammation and avoid prolonged inflammation. The 14:0 / 14:0 type phosphatidylserine (PS) in the phospholipid repair system can be directly integrated into the intestinal mucosal cell membrane of yellow catfish, specifically repair the tight junction protein ZO-1, reduce the intestinal permeability, and rebuild the intestinal physical barrier to enhance the ability to resist pathogenic invasion. Ethoxyquin can specifically scavenge hydroxyl radicals (OH) at the infection site, protecting the DNA of intestinal cells from oxidative damage; sophorose can inhibit the binding of Vibrio mimicus surface adhesion receptors (such as fucose residues) to intestinal epithelial cells by regulating the pH of the intestinal mucus layer, and at the same time optimize the intestinal microecological environment to assist in enhancing the barrier function. OH), protecting the DNA of intestinal cells from oxidative damage; sophorose can inhibit the binding of Vibrio mimicus surface adhesion receptors (such as fucose residues) to intestinal epithelial cells by regulating the pH of the intestinal mucus layer, and at the same time optimize the intestinal microecological environment to assist in enhancing the barrier function.

[0022] Through nanoliposome encapsulation technology, stable coexistence and targeted delivery of various components are achieved, avoiding inactivation of components in the gastrointestinal environment and ensuring that the various mechanisms work synergistically. DETAILED DESCRIPTION

[0023] The present application will be further described in conjunction with the following examples, but the embodiments of the present application are not limited thereto. If not specifically indicated, the technical means used in the following examples and experimental examples are conventional means known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels.

[0024] Example 1 Table 1 shows the efficacy components and the content and range of each component of a yellow catfish intestinal inflammation complex regulator containing ω-3 fatty acid-lipoxin-phospholipid.

[0025] Table 1: Formula components and proportions (efficacy components)

[0026]

[0027] Formula (1) In addition to the above-mentioned functional ingredients, lipid carriers, aqueous phase solvents and lyophilization protectants are also required. The raw materials of the lipid carriers include soybean phospholipids (meeting the requirements of "soybean phospholipids" in the fourth part of Chinese Pharmacopoeia 2025 Edition on pharmaceutical excipients, CAS No.: 8030-76-0) and cholesterol (CAS No.: 57-88-5); the aqueous phase solvent can be phosphate buffer (PBS); the lyophilization protectants include mannitol and trehalose.

[0028] The preparation of the complex control agent is carried out as follows: Step 1: Lipid film preparation The raw materials of the lipid carriers, eicosapentaenoic acid, lipoxin B4, phosphatidylserine and dehydroabietic acid in a mass ratio of 30-50:20-30:0.1-0.5:8-12:1-2 are added into a chloroform-methanol mixed solvent for mixing. Preferably, the mass ratio of the lipid carriers, eicosapentaenoic acid, lipoxin B4, phosphatidylserine and dehydroabietic acid is 40:25:0.3:10:1. Among them, the lipid carriers are composed of soybean phospholipids and cholesterol in a mass ratio of 60-80:20-40 (preferably 70:30); the amount ratio of the lipid carriers and the chloroform-methanol mixed solvent is 40-60 g:200-400 mL (preferably 50 g:300 mL), 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). 37℃ water bath magnetic stirring until completely dissolved to form a clear lipid solution.

[0029] The lipid solution is transferred to a rotary evaporator, and the solvent is removed by rotary evaporation under the condition of 60℃ and reduced pressure (≤50 mbar) until a uniform lipid film is formed on the inner wall of the flask; nitrogen blowing (50℃, 0.2 MPa pressure, for about 30 min) and ensuring that the residual amount of chloroform is ≤50 ppm. The flask is placed in a vacuum drying box (40℃, ≤10 mbar) for 2h to completely remove the residual solvent and obtain a drug-loaded lipid film.

[0030] Step 2: Preparation of aqueous phase Take pH 6.5 phosphate buffer (PBS), add sophorose and ethoxyquin, stir to dissolve, and then sterilize through a 0.22 μm filter membrane to obtain an aqueous solution. Among them, the amount ratio of sophorose, ethoxyquin and buffer is 10-18 g:0.01-0.03 g:300-500 mL (preferably 400 mL). Based on the mass of the lipid carrier (30-50 g), the volume to mass ratio of the aqueous phase to the lipid is controlled at 6-16.7 mL / g, preferably 10 mL / g, to ensure that the mass of the lipid film is about 1:10 to the volume of the aqueous phase, which is beneficial to the formation of uniform nanoliposomes. The overall material ratio is as follows: lipid carrier: eicosapentaenoic acid: lipoxin B4: phosphatidylserine: dehydroabietic acid sophorose: ethoxyquin: buffer = 30-50 g:20-30 g:0.1-0.5 g:8-12 g:1-2 g:10-18 g:0.01-0.03 g:300-500 mL. Preferably, the lipid carrier: eicosapentaenoic acid: lipoxin B4: phosphatidylserine: dehydroabietic acid sophorose: ethoxyquin: buffer = 40 g:25 g:0.3 g:10 g:1 g:15 g:0.02 g:400 mL.

[0031] Preheat the aqueous solution to 55°C, slowly add it to the flask containing the drug-loaded lipid film, and mix well to hydrate the lipid film.

[0032] Step 3: Ultrasonic dispersion to form nanoliposomes Use a vortex shaker to stir at low speed (200 rpm) to fully hydrate the lipid film to form a coarse emulsion. Transfer the coarse emulsion to an ultrasonic cell disruptor, set the power to 200 W and the frequency to 40 kHz, and ultrasonically treat it for 15 min according to the duty cycle "work for 3 seconds, intermittent for 3 seconds" until the emulsion becomes translucent. Pass through a lipid extruder (use a polycarbonate membrane with a pore size of 100 nm) to filter 3 times to obtain a uniform nanoliposome suspension with a particle size of 80-120 nm. The lipid extruder treatment can break the agglomeration of the original liposomes, allowing different sized liposomes in the coarse emulsion to pass through a polycarbonate membrane with a specific pore size, and ultimately obtaining a uniform system with a narrow particle size distribution. The extrusion process reshapes the lipid bilayer structure of the liposomes, reduces membrane defects, and makes the liposome shape more rounded and the membrane structure more stable.

[0033] Step 4: Freeze-drying molding To the nanoliposome suspension, add mannitol at a final concentration of 2-4% (preferably 3%, mass percentage) and trehalose at a final concentration of 1-3% (preferably 2%, mass percentage) as lyophilization protectants, mix well, and then distribute into a Schlenk flask. First, pre-freeze the Schlenk flask in a -40°C refrigerator for 2h to form a stable solid state. Then, perform vacuum freeze-drying, transfer to a freeze dryer, and dry at -40°C under a vacuum of <10 Pa for 12h, then increase the temperature to 25°C at a rate of 0.5°C / min, and after the temperature increase is complete, continue drying for 2h. After the above steps are completed, collect the finished product. The lyophilized preparation is in the form of a loose powder, which is sealed under nitrogen to prevent oxidation, thereby obtaining the ω-3 fatty acid-lipoxin-phospholipid-containing complex regulator of intestinal inflammation of yellow catfish according to the present solution.

[0034] For prophylactic purposes, the complex regulator prepared in Example 1 can be added to feed (e.g., Jiangtuan feed of Sichuan Zhongliangchuang Life Science and Technology Co., Ltd.) at a final concentration of 0.1-0.2% (mass percentage). Feed twice a day for 5 consecutive days, once a month, suitable for seedling stage and before and after transportation.

[0035] For therapeutic purposes, the complex regulator prepared in Example 1 can be added to feed at a final concentration of 0.3-0.4% (mass percentage). Feed three times a day for 10 consecutive days, combined with water disinfection (e.g., potassium peroxymonosulfate complex salt), suitable for acute inflammation.

[0036] Experimental Example Prepare the ω-3 fatty acid-lipoxin-phospholipid-containing complex regulator of intestinal inflammation of yellow catfish according to the formulations in Tables 2 and 3 for each experimental group and control group. The additives and other solvents (soybean phospholipids, cholesterol, chloroform-methanol mixed solvent, phosphate buffer, mannitol, and trehalose, etc.) are added according to the best mode of Example 1, which will not be repeated here.

[0037] Table 2: Formulation composition of active ingredients of experimental groups and control groups

[0038] Table 3: Formulation composition of active ingredients of experimental groups and control groups

[0039] The preparation methods of experimental groups 1-3 and control groups 1-9 are the same as the best mode of Example 1, and only need to omit the addition of a certain component in the corresponding step.

[0040] The formulation of control group 10 is the same as experimental group 1, except for the preparation method, which is as follows: The lipid carrier, eicosapentaenoic acid, lipoxin B4, phosphatidylserine, dehydroabietic acid, sophorobiose and ethoxyquinoline were added into chloroform-methanol mixed solvent, dissolved and rotary evaporated to obtain drug-loaded lipid film. Then PBS at 55°C was slowly added into the flask containing the drug-loaded lipid film, mixed uniformly to hydrate the lipid film. The nanoliposomes were formed by ultrasonic dispersion and extrusion. The nanoliposomes were freeze-dried to obtain loose powder of the complex regulator of fish intestinal inflammation containing omega-3 fatty acid-lipoxin-phospholipid. Except for the above-described operation mode, the rest of the operation mode, technical parameters and reagent dosage were completely the same as the best process mode of Example 1. That is, in the comparative group 10, all the six components were added into the lipid carrier.

[0041] The formula of the comparative group 11 was the same as that of the experimental group 1, except for the preparation method, which was as follows: Eicosapentaenoic acid, lipoxin B4 and phosphatidylserine were added into the lipid carrier to form a drug-loaded lipid film. In PBS, sophorobiose, ethoxyquinoline and dehydroabietic acid were added to form an aqueous solution. Except for the above-described operation mode, the rest of the operation mode, technical parameters and reagent dosage were completely the same as the best process mode of Example 1. That is, in the comparative group 11, dehydroabietic acid was added into the aqueous solution instead.

[0042] The formula of the comparative group 12 was the same as that of the experimental group 1, except for the preparation method, which was as follows: Eicosapentaenoic acid, lipoxin B4 and dehydroabietic acid were added into the lipid carrier to form a drug-loaded lipid film. In PBS, sophorobiose, ethoxyquinoline and phosphatidylserine were added to form an aqueous solution. Except for the above-described operation mode, the rest of the operation mode, technical parameters and reagent dosage were completely the same as the best process mode of Example 1. That is, in the comparative group 12, phosphatidylserine was added into the aqueous solution instead.

[0043] The formula of the comparative group 13 was the same as that of the experimental group 1, except for the preparation method, which was as follows: Eicosapentaenoic acid, lipoxin B4, dehydroabietic acid, phosphatidylserine and ethoxyquinoline were added into the lipid carrier to form a drug-loaded lipid film. In PBS, sophorobiose was added to form an aqueous solution. Except for the above-described operation mode, the rest of the operation mode, technical parameters and reagent dosage were completely the same as the best process mode of Example 1. That is, in the comparative group 13, ethoxyquinoline was added into the lipid instead.

[0044] The formulation of the comparative group 14 is the same as the experimental group 1, and the difference is the preparation method, which is as follows: the water phase solution is preheated to 50℃, slowly added to the flask containing the drug-loaded lipid membrane, and mixed uniformly to hydrate the lipid membrane. Except for the above-described operation mode, the rest of the operation mode, technical parameters, and reagent amount are exactly the same as the best process mode of Example 1. That is, in the comparative group 14, the preheating temperature of the water phase solution is reduced.

[0045] The formulation of the comparative group 15 is the same as the experimental group 1, and the difference is the preparation method, which is as follows: the water phase solution is preheated to 60℃, slowly added to the flask containing the drug-loaded lipid membrane, and mixed uniformly to hydrate the lipid membrane. Except for the above-described operation mode, the rest of the operation mode, technical parameters, and reagent amount are exactly the same as the best process mode of Example 1. That is, in the comparative group 15, the preheating temperature of the water phase solution is increased.

[0046] Taking the experimental group 1 as an example, the obtained nanoliposome suspension is detected, and the average of the nanoparticles is about 100 nm, and the PDI (polydispersity index) is <0.2. The EPA encapsulation rate is 88.5%, and the lipoxin B4 encapsulation rate is 92.3%. Using the lipid carrier to encapsulate EPA and lipoxin B4 helps the delivery and protection of these substances. After freeze-drying, the microbial indicators of the preparation are total bacterial count <100 CFU / g, mold and yeast <10 CFU / g, and pathogenic bacteria (Salmonella, Escherichia coli) are not detected.

[0047] The Ictalurus channel catfish intestinal inflammation complex regulating agents containing omega-3 fatty acids-lipoxin-phospholipids prepared by the experimental groups 1-3 and the comparative groups 1-15 are used to study the Vibrio clearance effect, the intestinal mucosa ZO-1 protein expression promotion, and the ROS clearance rate. The specific experimental method is as follows: The Ictalurus channel catfish with a body weight of 50±5 g is artificially infected with Vibrio mimicus (1×10 3 CFU / g body weight). After the fish is anesthetized, a sterile syringe is used to inject the bacterial solution from the abdominal cavity (the inner side of the base of the pectoral fin) to perform infection. Among them, Vibrio mimicus (1×10 Vibrio mimicusATCC 33653, the type strain of V. mimicus, which is commercially available). The intestinal inflammation complex regulator containing omega-3 fatty acid-lipoxin-phospholipid prepared in experimental groups 1-3 and comparative groups 1-15 was added into the feed, and the yellow-head catfish was fed. The adding amount of the complex regulator was 0.4% (added in the conventional Jiangtuan feed), and after the V. mimicus infection model was successfully established, the fish was fed with the feed containing the drug, 3 times a day. The total amount of the feed containing or not containing the complex regulator was 3% of the fish weight, and the single feeding amount was 1% of the fish weight. After continuous feeding for 5 days (detection was performed 5 days after infection), the indexes were detected. At the same time, a control group (fed with ordinary feed after infection) and a blank group (fed with ordinary feed without infection) were set.

[0048] The specific detection indexes are as follows: (1) Intestinal villus height detection (H&E staining method) The intestinal tissue of the experimental animal was quickly washed clean with normal saline to avoid residue of the contents. The tissue was cut into 0.5-1 cm small pieces and placed into 4% paraformaldehyde fixing solution for fixation for more than 24 h to ensure that the tissue was fully fixed. After dehydration and embedding, slicing and pasting, H&E staining, microscopic measurement was performed. The slices were observed under a microscope, 10 complete and clear intestinal villi of each sample were selected under 400 times magnification, and the height was measured by using the measurement software of the microscope, and the average value was taken as the intestinal villus height of the sample (single sample). The average value of 10 yellow-head catfish samples of each experimental group, comparative group, control group and blank group was taken as the measurement value of the experimental group.

[0049] This index reflects the intestinal mucosa structure integrity and nutritional absorption function state. The intestinal villus is a finger-like protrusion formed by the intestinal epithelium protruding into the intestinal cavity, and its height directly determines the absorption surface area of the intestinal mucosa, and the integrity of the villus morphology is the basis of the stability of the intestinal barrier structure. In the yellow-head catfish infection model, the invasion of pathogenic bacteria will destroy the intestinal epithelial cells, leading to damage such as atrophy, rupture and shedding of the intestinal villi, showing a decrease in villus height, and further causing a decrease in nutritional absorption efficiency and an increase in intestinal barrier permeability. This index quantifies the intestinal villus morphology parameters, objectively evaluates the protection or repair effect of the complex regulator on the intestinal mucosa structure, and the detection result can be used as the morphological evidence of the intestinal mucosa protection effect of the complex regulator, to prove the effect of the complex regulator on improving the intestinal structure damage caused by infection.

[0050] (2) Intestinal mucosa ZO-1 protein expression amount detection (ELISA method) Take the intestinal tissue 5 days after infection, extract total protein, determine the protein concentration with BCA kit, adjust the total protein concentration to 1 mg / mL. ZO-1 protein ELISA kit (according to the kit instructions) for detection, enzyme labeling instrument at 450 nm wavelength to determine the absorbance (OD value) of each well, according to the standard curve of standard product OD value, calculate the ZO-1 protein concentration in the sample; with the control group protein expression as the reference (set as 1.00), calculate the relative expression value of each experimental group, comparison group, control group.

[0051] This index reflects the integrity of intestinal epithelial tight junction (core index of intestinal barrier function). ZO-1 (occludin 1) is a key structural protein of the tight junction between intestinal epithelial cells, and its expression is directly related to the stability of the tight junction: as the core of the physical barrier of the intestinal barrier, the tight junction can prevent pathogenic bacteria, toxins and undigested macromolecules in the intestine from penetrating the epithelium into the circulatory system, maintaining the intestinal homeostasis. In the state of infection, the virulence factors of pathogenic bacteria will degrade ZO-1 protein, leading to loose tight junction and increased intestinal permeability, and thus aggravating the infection damage. This index quantifies the functional status of the intestinal barrier by detecting the relative expression of ZO-1 protein. If the relative expression value of ZO-1 in the complex regulator intervention group is significantly higher than that in the infection control group, it indicates that the complex regulator can enhance the mechanical barrier function of the intestinal epithelium by up-regulating the expression of tight junction protein, reduce the transmembrane transport of pathogenic bacteria and their metabolites, provide molecular level evidence for the technical effect of the complex regulator on improving the permeability of the intestinal barrier, and provide supporting data for the complex regulator to enhance the defense function of the intestine.

[0052] (3) Number of Vibrio in the intestine (plate counting method) Take 0.5g of intestinal tissue 5 days after infection to make homogenate, and perform gradient dilution. Take 0.1mL of bacterial solution of different dilutions, spread on TCBS medium plate, and incubate at 28℃ for 24h. Select the plates with 30-300 colonies for counting, and calculate the Vibrio load in each gram of intestinal tissue (CFU / g).

[0053] This index reflects the proliferation ability of pathogenic bacteria in the intestine and the antibacterial effect of the complex regulator. As the pathogenic bacteria of the infection model, the load of Vibrio in the intestine (CFU / g) 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, and by gradient dilution and counting, the number of surviving Vibrio in the intestinal tissue can be accurately quantified, which is a functional index for evaluating the direct antibacterial effect or indirect inhibition of pathogenic bacteria proliferation of the complex regulator. The detection result of this index is the direct evidence of the technical effect of the complex regulator on anti-intestinal pathogenic bacteria infection.

[0054] (4) Intestinal tissue interleukin-6 (IL-6) content detection (ELISA method) Take the intestinal tissue of yellow-head catfish (about 0.1 g) 5 days after infection, rinse it clean in pre-cooled physiological saline, and then weigh it after absorbing it with filter paper. Put the tissue in a glass homogenizer, add pre-cooled PBS (pH 7.2-7.4) at a weight-to-volume ratio of 1:9 for ice bath mechanical homogenization. Transfer the homogenate to a centrifuge tube and centrifuge at 5000 rpm for 10 min at 4°C. Carefully collect the supernatant and store it at -80°C for testing. Use the fish-specific interleukin-6 (IL-6) ELISA detection kit (e.g., Shanghai Zymed Biological Technology Co., Ltd., Item No.: ML063133F) and strictly follow the kit instructions to operate. The brief steps are as follows: add the standard and tissue homogenate supernatant to the pre-coated antibody enzyme-labeled plate holes, incubate, and then wash; add biotinylated detection antibody, incubate again, and then wash; add horseradish peroxidase (HRP)-labeled streptavidin, incubate, and then wash thoroughly; add substrate TMB for color development reaction, and finally add stop solution to stop the reaction. Immediately measure the absorbance (OD value) of each hole at 450 nm wavelength with an enzyme-labeled instrument. According to the standard concentration and the corresponding OD value, draw a standard curve, and put the OD value of the sample into the standard curve to calculate the IL-6 content in the intestinal tissue per unit weight (unit: pg / g). At the same time, use the BCA method to determine the total protein concentration of each sample, and the final result can also be corrected to pg / mg prot to eliminate the error caused by the difference in protein concentration.

[0055] This index reflects the intensity and degree of local intestinal inflammatory response. IL-6 is a typical pro-inflammatory cytokine. After pathogenic bacteria infection, intestinal mucosal immune cells (such as macrophages and lymphocytes) are activated and secrete a large amount of IL-6, and 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 destroy intestinal mucosal homeostasis; and moderate regulation of inflammatory response is the key to reducing tissue damage caused by infection.

[0056] This index quantifies the absolute content of IL-6 in intestinal tissue to evaluate the anti-inflammatory activity of the complex regulator: if the IL-6 content of the complex regulator intervention group is significantly lower than that of the infection control group, it indicates that the complex regulator can regulate the intensity of local intestinal inflammation by inhibiting the secretion of pro-inflammatory cytokines, reduce the damage of inflammatory factors to the intestinal mucosa, and achieve the effect of anti-inflammatory and intestinal protection. The detection result provides immunological evidence at the molecular level for the technical effect of the complex regulator in improving intestinal inflammation caused by infection.

[0057] (5) Intestinal tissue superoxide dismutase (SOD) activity and malondialdehyde (MDA) content detection Intestinal tissue (about 0.1 g) from the same part of the fish at 5 days after infection was rinsed clean in pre-cooled normal saline and weighed after being absorbed with filter paper. The tissue was placed in a glass homogenizer and pre-cooled normal saline was added at a weight-to-volume ratio of 1:9 for ice-bath mechanical homogenization to prepare 10% tissue homogenate. The homogenate was centrifuged at 4°C and 2500-3000 rpm for 10-15 min, and the supernatant was carefully collected for use.

[0058] SOD activity determination: The xanthine oxidase method (hydroxylamine method) SOD determination kit was used for operation. The main principle is that SOD inhibits the oxidation of hydroxylamine by superoxide anion free radicals (O2· - ), and the activity of SOD is calculated by measuring the absorbance value of the reaction system. The steps were strictly followed according to the kit instructions, and the spectrophotometer was used to measure the absorbance (OD value) at 550 nm wavelength. According to the formula, the SOD activity in intestinal tissue per unit weight was calculated, expressed as U / mg prot.

[0059] MDA content determination: The thiobarbituric acid (TBA) method MDA determination kit was used for operation. The main principle is that MDA condenses with TBA under acidic conditions to generate a red product, which has a maximum absorption peak at 532 nm wavelength. The steps were strictly followed according to the kit instructions, and the spectrophotometer was used to measure the absorbance (OD value) at 532 nm wavelength. According to the formula, the MDA content in intestinal tissue per unit weight was calculated, expressed as nmol / mg prot.

[0060] The index reflects the oxidative stress state and antioxidant damage capacity of intestinal tissue. SOD is an important endogenous antioxidant enzyme in the body, and its core function is to catalyze the dismutation of superoxide anion free radicals into hydrogen peroxide, which is then removed by other antioxidant systems, reducing the oxidative damage of free radicals to cell lipids, proteins and nucleic acids, and is a key enzyme for maintaining the balance of oxidation-antioxidation in the body. In the infection model, the invasion of pathogenic bacteria will trigger the oxidative stress response of intestinal tissue, leading to the accumulation of a large number of free radicals, and the activity of SOD will decrease due to consumption. The activity level of this index directly reflects the antioxidant capacity of intestinal tissue: the SOD activity of the drug intervention group is significantly higher than that of the infection control group, indicating that the drug can enhance the activity of endogenous antioxidant enzymes and improve the ability of intestinal tissue to remove free radicals, reducing the damage of oxidative stress to intestinal epithelial cells. MDA is the end product of lipid peroxidation reaction, and its content is positively correlated with the degree of cell oxidative damage: when free radicals attack unsaturated fatty acids on the cell membrane, they will trigger a chain reaction of lipid peroxidation, leading to cell membrane structure damage and a large amount of MDA generation and accumulation. The content level of this index directly reflects the degree of oxidative damage of intestinal tissue: the MDA content of the drug intervention group is significantly lower than that of the infection control group, indicating that the drug can inhibit the occurrence of lipid peroxidation reaction and reduce the accumulation of oxidative damage products, protecting the integrity of intestinal cell membrane.

[0061] 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.

[0062] In summary, the above indicators construct a multi-target evaluation system for the effect of drugs on infectious intestinal injury from five dimensions of structure and morphology (intestinal villus height), barrier function (ZO-1), pathogenic bacteria clearance (vibrio load), inflammation regulation (IL-6), and oxidative damage protection (SOD / MDA). The experimental results are shown in Tables 4 and 5.

[0063] Table 4: Detection results of each index after 5 days of infection (blank group, experimental group, comparison groups 1-6; mean ± standard deviation, n=10)

[0064] Table 5: Detection results of each index after 5 days of infection (control group, comparison groups 7-15; mean ± standard deviation, n=10)

[0065] (1) The overall effect of the ω-3 fatty acid-lipoxin-phospholipid-containing compound for regulating and controlling the inflammation of Ictalurus funnalis intestinal tract in the scheme The experimental groups 1-3 are the embodiments of the scheme, the efficacy ingredients of which are all within the preferred range, and all the detection indexes show ideal effects, and are significantly better than the subsequent comparison groups and the control group, fully verifying the scientificity and feasibility of the formula system of the scheme.

[0066] Compared with the blank group and the control group, the composite intestinal health regulator prepared by the experimental groups 1-3 has the following effects: Intestinal structure protection: The intestinal villus height of the experimental groups 1-3 is 681.5±11.7 μm, 654.8±14.9 μm and 667.6±13.8 μm respectively, which is lower than that of the uninfected blank group (753.8±8.2 μm), but is much higher than that of the infected control group (476.8±11.3 μm), and is significantly better than that of most comparison groups, indicating that the preparation can effectively reduce the intestinal villus atrophy and rupture caused by Vibrio infection, and maintain the integrity of the intestinal mucosa structure.

[0067] Barrier function enhancement: The relative expression amount of ZO-1 protein is 1.823±0.148, 1.701±0.121 and 1.752±0.139 respectively, which is significantly higher than that of the control group, proving that the preparation can effectively up-regulate the expression of intestinal tight junction protein, strengthen the mechanical barrier function of the intestine, and reduce the transmembrane transport of pathogenic bacteria.

[0068] Significant bacteriostatic effect: The Vibrio load in the intestine is 1.52±0.31×10 5 CFU / g, 2.08±0.39×10 5 CFU / g and 1.79±0.32×10 5 CFU / g respectively, which is reduced by 54.8%-67.6% compared with the control group (4.69±0.51×10 5 CFU / g), indicating that the preparation can effectively inhibit the colonization and proliferation of Vibrio in the intestine.

[0069] Prominent anti-inflammatory effect: The IL-6 content in the intestine is 38.45±4.15 pg / mg prot, 45.18±5.03 pg / mg prot and 41.76±4.42 pg / mg prot respectively, which is significantly lower than that of the control group (95.75±10.15 pg / mg prot), and is close to the level of the blank group (25.10±2.75 pg / mg prot), indicating that the preparation can effectively regulate the local inflammatory response in the intestine and reduce the damage of inflammatory factors to the mucosa.

[0070] The anti-oxidative damage ability is excellent: the SOD activity of the experimental groups 1-3 (78.6-85.2 U / mg prot) is 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) is 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-antioxidation balance.

[0071] Therefore, the formula dosage range of the experimental groups 1-3 (EPA 20-30 parts, lipoxin B4 0.1-0.5 parts, PS 8-12 parts, dehydroabietic acid 1-2 parts, sophorabiose 10-18 parts, and ethoxyquinoline 0.01-0.03 parts) is scientific and reasonable, and excellent synergistic effects are exhibited in intestinal structure protection, barrier function enhancement, antibacterial, anti-inflammatory, antioxidant, and the like.

[0072] (2) Analysis of the synergistic effect of EPA, lipoxin B4, and PS Necessity of the combination of the three The comparative groups 1-3 respectively lack EPA, PS, and lipoxin B4, and the rest of the components and preparation process are consistent with those of the experimental group 1. The differences in the index performance of the experimental group 1 directly reflect the influence of the lack of a single component on the overall effect. Compared with the experimental group 1, the index performance of the three after the lack of the three appears significant deterioration. The indexes of the comparative groups 1-3 are significantly worse than those of the experimental group 1. After the lack of any component, the function of the preparation is greatly reduced, among which the influence of the lack of EPA is the most significant, proving that EPA, lipoxin B4, and PS are interdependent in function and indispensable, and are the key combination for the preparation to play the core effect.

[0073] Verification of the intensification of the synergistic effect of the three The comparative groups 4-6 respectively lack two components, and the dosages of the rest of the components are consistent with those of the experimental group 1: the comparative group 4 is EPA alone, the comparative group 5 is PS alone, and the comparative group 6 is lipoxin B4 alone. The detection results show that after the lack of two components, the index deterioration degree is more significant than that of the lack of a single component.

[0074] The synergistic effect of the three compounds for six technical indicators was calculated, and the synergistic coefficient was used to represent it. Synergistic coefficient = measured joint effect value / theoretical joint effect value; synergistic coefficient > 1, synergistic effect; synergistic coefficient = 1, additive effect, synergistic coefficient < 1, antagonistic effect. According to the Bliss model, the theoretical joint 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); A represents EPA, B represents PS, and C represents lipoxin B4. For the indicators of intestinal villus height, ZO-1 relative expression amount, and SOD activity (the higher the value, the better the effect), the calculation method of E(A) or E(B) or E(C) or E measured (measured joint effect value) is: (sample value of comparison group 4 or 5 or 6 or experimental group 1 - control group value) / (blank group value - control group value) × 100%. For the indicators of intestinal Vibrio number, IL-6 content, and MDA content (the lower the value, the better the effect), the calculation method of E(A) or E(B) or E(C) or E measured (measured joint effect value) is: (control group value - sample value of comparison group 4 or 5 or 6 or experimental group 1) / (control group value - blank group value) × 100%. The synergistic coefficient is calculated for the above six indicators, and the calculation results are all > 1, among which the numerical values of the synergistic coefficients of the indicators of intestinal Vibrio number, ZO-1 relative expression amount, and IL-6 content are more significant (about 1.7, about 1.5, and about 1.2).

[0075] Therefore, it can be seen that EPA, PS, and lipoxin B4 jointly up-regulate the expression of tight junction proteins, strengthen the intestinal mechanical barrier; make up for the insufficient antibacterial effect of single EPA, inhibit the colonization and proliferation of Vibrio through combined action; synergistically inhibit the secretion of pro-inflammatory factors, regulate the intensity of local intestinal inflammation; synergistically inhibit lipid peroxidation, reduce the accumulation of oxidative damage products; jointly maintain the integrity of intestinal mucosal structure, reduce the atrophy of villi caused by Vibrio infection; synergistically enhance the activity of antioxidant enzymes, and assist in removing free radicals in the intestine. The three compounds have synergistic effects on five main target points of intestinal barrier function strengthening (ZO-1), pathogenic bacteria inhibition (Vibrio number), inflammation regulation (IL-6), oxidative damage protection (MDA / SOD), and mucosal structure protection (intestinal villus height), forming a multi-target synergistic regulation system. The effect of single component or binary combination is significantly worse than that of ternary combination (synergistic coefficient > 1), which proves that the technical scheme of the combination of the three is not a simple superposition, but achieves effect amplification through synergistic effect, and obtains unexpected technical effect.

[0076] (3) Effect analysis of the combination of sophorobiose and ethoxyquin Sophorose and ethoxyquin are antioxidants in the prior art, and their combination use produces a variety of synergistic effects in the use environment of the present scheme, as shown in the experimental data of comparative groups 7 and 8. According to the Bliss model, the theoretical combined effect value of the two factors is: Etheoretical = E(A) + E(B) - E(A)E(B); A represents sophorose, and B represents ethoxyquin. The synergistic coefficient is calculated for the above six indicators, and the calculation results are all >1, among which the values of the synergistic coefficients of the indicators of the number of Vibrio and the content of IL-6 are more significant (about 1.1). Sophorose and ethoxyquin have synergistic effects in the six indicators, among which the inflammation regulation (IL-6) is a significant synergy, forming a multi-dimensional synergistic system of anti-oxidation, anti-inflammation, barrier protection and bacteriostasis. Based on the complementary characteristics, they respectively act on different target points such as oxidative stress, inflammatory response and intestinal structure, and their synergistic effect is not a simple superposition of the effects of single components, but an effect amplification through functional complementation, and an unexpected technical effect is obtained.

[0077] (4) Effect analysis of dehydroabietic acid Comparative group 9 lacks dehydroabietic acid, and the dosages and preparation processes of the other components are consistent with those of experimental group 1, and the differences in the indicators between them directly reflect the function of dehydroabietic acid: In terms of intestinal villus height, the comparative group 9 is 557.2±19.8 μm, which is 18.2% lower than 681.5±11.7 μm of the experimental group 1, and the intestinal mucosa damage is more serious; the relative expression amount of ZO-1 is 1.198±0.097, which is 34.3% lower than that of the experimental group 1, and the intestinal barrier permeability is increased; the Vibrio load is 4.03±0.49×10 5 CFU / g, which is 165.1% higher than that of the experimental group 1, and the pathogenic bacteria proliferation ability is greatly enhanced; the IL-6 content is 75.14±8.05 pg / mg prot, which is 95.4% higher than that of the experimental group 1, and the inflammatory response is intensified; the SOD activity is 62.8±7.8 U / mg prot, which is 26.3% lower than that of the experimental group 1, and the antioxidant enzyme activity is consumedly decreased; the MDA content is 2.73±0.49 nmol / mg prot, which is 125.6% higher than that of the experimental group 1, and the lipid peroxidation product is accumulated, and the cell damage is more serious.

[0078] The core function of dehydroabietic acid is to directly inhibit the proliferation of Vibrio in the intestine, and in the present scheme, it produces an enhancing effect in the aspects of intestinal barrier function enhancement (ZO-1), inflammation regulation (IL-6), oxidative damage protection (MDA / SOD) and mucosa structure protection (intestinal villus height) in addition to its function as a bacteriostatic agent.

[0079] (5) Effect of preparation process on the effect of the preparation The formulation of comparative group 10-15 is completely consistent with experimental group 1, only the single preparation process parameter is different, the specific difference and index performance are as follows: Key role of component distribution mode (comparative groups 10-13) The optimized distribution scheme of experimental group 1 is: EPA, lipoxin B4, PS, dehydroabietic acid are added to the lipid phase, sophorose, ethoxyquinoline are added to the water phase. The component distribution mode of comparative groups 10-13 is different: Comparative group 10 adds all components (including sophorose, ethoxyquinoline) to the lipid phase, and the rest of the process parameters are consistent with experimental group 1. The detection results show that it performs the worst: the intestinal villus height is 519.4±24.6μm, which is 23.8% lower than experimental group 1; the relative expression amount of ZO-1 is 1.047±0.079, which is reduced by 42.6%; the intestinal Vibrio load is 4.79±0.58×10 5 CFU / g, increased by 215.1%; IL-6 content is 82.36±8.83 pg / mg prot, increased by 114.2%; SOD activity is 58.5±8.7 U / mg prot, decreased by 31.3%; MDA content is 3.02±0.57 nmol / mg prot, increased by 149.6%.

[0080] Comparative group 11 changes dehydroabietic acid to be added to the water phase, and the rest of the process is consistent with experimental group 1. The index performance is: the intestinal villus height is 634.5±14.9μm, which is 6.9% lower than experimental group 1; the relative expression amount of ZO-1 is 1.551±0.128, which is reduced by 14.9%; the intestinal Vibrio load is 1.98±0.29×10 5 CFU / g, increased by 30.3%; IL-6 content is 58.61±6.24 pg / mg prot, increased by 52.4%; SOD activity is 73.9±5.9 U / mg prot, decreased by 13.3%; MDA content is 1.69±0.35 nmol / mg prot, increased by 39.7%.

[0081] Comparative group 12 changes PS to be added to the water phase, and the rest of the process is consistent with experimental group 1. The index performance is: the intestinal villus height is 544.3±21.7μm, which is 20.1% lower than experimental group 1; the relative expression amount of ZO-1 is 1.146±0.091, which is reduced by 37.1%; the intestinal Vibrio load is 3.76±0.48×10 5 CFU / g, increased by 147.4%; IL-6 content is 73.79±7.85 pg / mg prot, increased by 91.9%; SOD activity is 64.2±7.7 U / mg prot, decreased by 24.6%; MDA content is 2.41±0.48 nmol / mg prot, increased by 99.2%.

[0082] Comparative group 13 changed ethoxyquin to add to the lipid phase, and the rest of the process was consistent with experimental group 1. The indicators were as follows: intestinal villus height 614.6 ± 16.8 μm, 9.8% lower than experimental group 1; ZO-1 relative expression 1.576 ± 0.121, 13.5% lower; intestinal Vibrio load 1.91 ± 0.28 × 10 5 CFU / g, increased by 25.7%; IL-6 content 52.37 ± 5.58 pg / mg prot, increased by 36.2%; SOD activity 76.6 ± 5.2 U / mg prot, decreased by 10.1%; MDA content 1.62 ± 0.29 nmol / mg prot, increased by 33.9%.

[0083] Optimization range of hydration temperature (comparative groups 14-15) The water phase preheating temperature of experimental group 1 was 55°C, the water phase preheating temperature of comparative group 14 was reduced to 50°C, and the water phase preheating temperature of comparative group 15 was increased to 60°C, and the rest of the process parameters were consistent with experimental group 1: The indicators of comparative group 14 (50°C) were as follows: intestinal villus height 647.7 ± 13.6 μm, 4.9% lower than experimental group 1; ZO-1 relative expression 1.781 ± 0.137, 2.3% lower; intestinal Vibrio load 1.68 ± 0.27 × 10 5 CFU / g, increased by 10.5%; IL-6 content 41.15 ± 4.38 pg / mg prot, increased by 7.0%; SOD activity 83.8 ± 4.9 U / mg prot, decreased by 1.6%; MDA content 1.38 ± 0.28 nmol / mg prot, increased by 14.0%. 50°C still met the hydration needs of the lipid membrane, only slightly affected the uniformity of liposome formation, had little effect on the overall effect, and belonged to an acceptable range.

[0084] The indicators of comparative group 15 (60°C) were as follows: intestinal villus height 644.8 ± 14.7 μm, 5.4% lower than experimental group 1; ZO-1 relative expression 1.749 ± 0.129, 4.1% lower; intestinal Vibrio load 1.81 ± 0.29 × 10 5 CFU / g, increased by 19.1%; IL-6 content 43.76 ± 4.65 pg / mg prot, increased by 13.8%; SOD activity 81.5 ± 5.1 U / mg prot, decreased by 4.3%; MDA content 1.51 ± 0.30 nmol / mg prot, increased by 24.8%. 60°C did not cause lipid membrane degradation, only a small amount of ingredients showed decreased stability due to the high temperature, and the effect was slightly lower than experimental group 1, but still significantly better than other process abnormal groups.

[0085] In summary, EPA, lipoxin B4, PS, dehydroabietic acid must be added to the lipid phase, sophorose, ethoxyquin must be added to the aqueous phase, this distribution mode can improve the stability and bioavailability of the components, which is the premise of the preparation to play the effect. The preheating temperature of the aqueous phase should be controlled at 50-60℃, preferably 55℃, this temperature range can ensure that the lipid film is fully hydrated, forming nano-liposomes with uniform particle size (80-120nm) and stable structure, avoiding insufficient hydration caused by too low temperature or component degradation caused by too high temperature.

[0086] (6) Summary This experiment verifies the technical advantages of the composite regulator through multiple control designs from the aspects of formula synergy, key component function and preparation process: Formula synergy effect: EPA, lipoxin B4 and PS are indispensable, and they can regulate intestinal barrier function and inflammatory response through synergistic effect; sophorose and ethoxyquin can reduce oxidative stress damage through antioxidant synergistic effect; dehydroabietic acid as the core antibacterial component forms a synergistic system of antibacterial, defense and repair with the above components.

[0087] Key component function: dehydroabietic acid is the direct antibacterial core, EPA is the synergistic regulation core, PS is the barrier function enhancement core, lipoxin B4 is the anti-inflammatory core, sophorose and ethoxyquin are the antioxidant core, each component plays its own role and cooperates with each other.

[0088] Process optimization key: the accurate distribution of components in the lipid phase and aqueous phase and the hydration temperature of about 55℃ are the core process parameters to ensure the effect of the preparation, which directly affects the encapsulation rate, stability and bioavailability of the components.

[0089] The above is only an embodiment of the present application, and the specific structure and properties of the known scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope of this application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain 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 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.

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 include: 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. 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 lipid carrier is composed of soybean lecithin and cholesterol in a mass ratio of 60-80:20-40; 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 preheating the aqueous solution to 55°C, lipid membrane hydration was 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 with a final concentration of 2-4% and trehalose with 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 application of the compound regulatory agent for intestinal inflammation of yellow catfish containing ω-3 fatty acids-lipoxygen-phospholipids as described in claim 9 in the preparation of a drug for relieving intestinal inflammation of yellow catfish caused by Vibrio mimicry infection, or in the preparation of a drug for inhibiting Vibrio mimicry infection.

Images