Application of fructus terminaliae billericae in relieving fish food-borne intestinal inflammation, regulating intestinal flora and inhibiting bacteria

Abstract

The invention discloses application of fructus terminaliae billericae in relieving fish food-borne intestinal inflammation, regulating intestinal flora and inhibiting bacteria, based on a 9-day soybean meal induced zebra fish enteritis model, 1% o of fructus terminaliae billericae is added, so that aggregation of neutrophils and macrophages in middle and rear intestines can be relieved, and zebra fish food-borne intestinal inflammation can be relieved; meanwhile, a 16S rRNA (ribosomal Ribonucleic Acid) gene sequencing result shows that the intestinal flora homeostasis of the fish with the food-borne intestinal inflammation can be regulated by adding 0.1% of the fructus terminaliae billericae; an aeromonas hydrophila in-vitro bacteriostasis experiment shows that the fructus terminaliae billericae aqueous extract has a remarkable bacteriostasis effect on aeromonas hydrophila, and a zebra fish in-vitro toxicity attacking experiment shows that the survival rate of zebra fish can be increased by adding 0.1% of fructus terminaliae billericae.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture drugs, and particularly relates to application of Terminalia chebula in preparing drugs for alleviating food-borne intestinal inflammation in fish, regulating intestinal flora and inhibiting bacteria. Background Art

[0002] Aquatic products are an important source of protein for humans. China ranks among the top in the world in terms of aquaculture output and economic benefits. As my country's aquaculture industry continues to grow and develop, some economic obstacles have emerged that affect the aquaculture industry. The most significant of these obstacles is the initial large-scale use of antibiotics in response to aquatic diseases such as bacterial and viral infections. Although some results were achieved in the initial disease control, the overuse of antibiotics has resulted in antibiotic residues being detected in many aquatic products, which is detrimental to human health. Therefore, people are actively researching alternatives to antibiotics in the field of aquatic disease prevention and control. Studies have found that many Chinese herbal extracts have a good effect on the prevention and treatment of aquatic diseases, and the treatment process is green and pollution-free, making them a good alternative to antibiotics for disease prevention and control. Since then, the application of Chinese herbal preparations in the aquatic industry has made great progress.

[0003] Aeromonas hydrophila is a Gram-negative bacterium that is widely distributed in various water bodies. It is a pathogen of human-animal-fish comorbidities, which can cause aquatic organisms to suffer from diseases such as sepsis, diarrhea, and enteritis, causing great losses to the aquatic industry and seriously affecting its development.

[0004] Terminalia chebula (Herba chebula) is a representative Tibetan medicine. It is the dried mature fruit of Terminalia chebula (Terminalia Linn), a plant of the Combretaceae family. It is primarily produced in South and Southeast Asia, with smaller quantities found in Tibet and Yunnan, my country. Li Shizhen noted in his Compendium of Materia Medica that Terminalia chebula can "relieve qi and stop diarrhea." Traditional Chinese Medicine considers Terminalia chebula to be neutral in nature, sweet, and astringent in taste, and primarily used to clear heat and detoxify, astringe and nourish the blood, and harmonize various medicinal herbs. It is used to treat various heat syndromes, diarrhea, liver and gallbladder disorders, and post-illness weakness. Modern medical research has also shown that its pitted fruit, often used as a traditional Tibetan medicinal ingredient, has the effects of clearing heat and detoxifying, astringing and nourishing the blood, and harmonizing various medicinal herbs. It also possesses multiple pharmacological activities, including anticancer, antioxidant, antibacterial, anticonvulsant, liver-protective, cholesterol-lowering, blood-sugar-lowering, and myocardial necrosis prevention. Currently, there are only reports that Terminalia chebula extract and Terminalia chebula polyphenols can inhibit the maturation of dendritic cells, effectively alleviate inflammatory responses, and have the effect of treating mouse enteritis (CN111973638A). In a mouse enteritis (IBD) model induced by the chemical medium dextran sulfate sodium (DSS), they have an inhibitory effect on the migration of bone marrow-derived dendritic cells, while their effect on intestinal mucosal immune cells in fish food-borne intestinal inflammation is unknown. Summary of the Invention

[0005] The object of the present invention is to provide new uses of Terminalia chebula Retz., including applications in the preparation of drugs for inhibiting Aeromonas hydrophila infection in fish, applications in the preparation of drugs for alleviating foodborne intestinal inflammation in fish, and applications in the preparation of products for improving the intestinal flora of fish with foodborne intestinal inflammation.

[0006] To achieve the above object, the present invention adopts the following technical measures:

[0007] Application of Terminalia chebula Retz. in the preparation of drugs for alleviating foodborne intestinal inflammation in fish: In a specific embodiment of the present invention, a foodborne enteritis model was established by feeding juvenile zebrafish with a feed in which 50% of the protein source was replaced by soybean meal. Terminalia chebula Retz. was added at 0.5‰, 1‰, and 2‰ respectively to the feed in which 50% of the protein source was replaced by soybean meal. Imaging analysis of innate immune cells of 9 dpf juvenile zebrafish was performed to examine the effect of improving inflammation. The results showed that, compared with the group fed with the feed in which 50% of the protein source was replaced by soybean meal, adding Terminalia chebula Retz. to the soybean meal-based feed could alleviate the aggregation of neutrophils and macrophages in the mid and hindgut to a certain extent, and the effect of 1‰ Terminalia chebula Retz. in alleviating the aggregation of neutrophils and macrophages was the best. Therefore, Terminalia chebula Retz. has a relieving effect on foodborne intestinal inflammation in fish.

[0008] Application of Terminalia chebula Retz. in the preparation of products for improving the intestinal flora of fish with foodborne intestinal inflammation: In a specific embodiment of the present invention, after feeding 3-month-old zebrafish with a protein source feed, a feed in which 50% of the protein source was replaced by soybean meal, and a 50% soybean meal replacement protein source feed containing 1‰ Terminalia chebula Retz. for 2 weeks, random samples of the whole intestine of each group were taken for 16S rRNA gene sequencing to determine the regulatory effect of Terminalia chebula Retz. on the intestinal flora. The results showed that adding Terminalia chebula Retz. could regulate the intestinal flora of fish with foodborne intestinal inflammation. At the genus level, the abundances of Faecalibacterium, Bifidobacterium, and Bacteroides were increased, improving the intestinal homeostasis of fish with foodborne intestinal inflammation.

[0009] Application of Terminalia chebula Retz. in the preparation of drugs for inhibiting Aeromonas hydrophila infection in fish: In a specific embodiment of the present invention, the antibacterial effect of the water extract of Terminalia chebula Retz. on Aeromonas hydrophila was preliminarily verified by measuring the inhibition zone. The minimum inhibitory concentration of Terminalia chebula Retz. against Aeromonas hydrophila was 2.5 mg / ml, and the minimum bactericidal concentration was 12.5 mg / ml. Further, after feeding 3-month-old zebrafish with a 50% soybean meal replacement protein source feed containing 1‰ Terminalia chebula Retz. for 6 weeks, a 10-day Aeromonas hydrophila challenge experiment was carried out to measure the in vivo anti-Aeromonas hydrophila ability of Terminalia chebula Retz. on zebrafish. The results showed that 1‰ Terminalia chebula Retz. could significantly improve the survival rate of zebrafish under Aeromonas hydrophila infection.

[0010] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0011] By establishing a foodborne enteritis model induced by soybean meal, it was found that compared with the soybean meal group, the aggregation of neutrophils and macrophages in the mid and hindgut of the Terminalia chebula Retz. group was significantly reduced, reflecting the decrease in its inflammation level, and confirming the effect of Terminalia chebula Retz. in alleviating foodborne enteritis. At the same time, the results of 16S rRNA gene sequencing showed that after adding Terminalia chebula Retz., the richness and diversity of the intestinal flora were higher than those in the soybean meal group, significantly improving the steady-state level of the intestinal flora.

[0012] The present invention first proposes the application of Terminalia chebula Retz. in inhibiting Aeromonas hydrophila, providing a new alternative antibiotic feed additive for further preventing the infection of fish-related pathogenic bacteria. The Terminalia chebula Retz. used is a natural medicinal plant, which has the advantages of being green, environmentally friendly, safe, pollution-free, effective, and low-cost as a feed additive ingredient. Brief Description of the Drawings

[0013] Figure 1 It is a fluorescence imaging result diagram of neutrophils and macrophages in the intestine of zebrafish larvae at 9 dpf after adding Terminalia chebula Retz. to the soybean meal feed.

[0014] Figure 2 It is the relative abundance at the phylum level of 16S rRNA gene sequencing in the intestines of different feed groups.

[0015] Figure 3 It is the relative abundance at the genus level of 16S rRNA gene sequencing in the intestines of different feed groups.

[0016] Figure 4 It is the Shannon-Winener index curve indicating the diversity of the flora. The ordinate is the Shannon-Winener index, and the abscissa is the number of sampled sequences.

[0017] Figure 5 It is the bacteriostatic circle diagram of Terminalia chebula Retz. against Aeromonas hydrophila.

[0018] Figure 6 It is the growth curve of the co-culture of Terminalia chebula Retz. and Aeromonas hydrophila.

[0019] Figure 7 It is the survival rate of each group after Aeromonas hydrophila challenge. Detailed Embodiments

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation to the present invention. All molecular biology operation methods involved in the embodiments are conventional methods well-known to those skilled in the art unless otherwise specified.

[0021] The following are the test materials, reagents, and instrument equipment involved in the embodiments:

[0022] 1. Biological materials: The wild-type AB zebrafish (Danio rerio) and transgenic zebrafish lines used in this experiment, including Tg(lyz:DsRED2) and Tg(mpeg1:EGFP), were all purchased from the China Zebrafish Resource Center (http: / / zfish.cn / ). The Tg(lyz:DsRED2); Tg(mpeg1:EGFP) double-fluorescent line was obtained by crossing two single-color fluorescent lines. The strains used in this invention were provided by the National Genebank of Aquatic Organisms - Sub-bank of Special Aquatic Animal Germplasm Resources. Among them, Aeromonas hydrophila was coded as Aer-028 and was isolated from the liver of grass carp in 2008.

[0023] 2. Reagents and consumables: Terminalia chebula (purchased from Shandu), low melting point agarose (UltraPureTM LMP Agarose, invitrogen), 4% paraformaldehyde (barsharp), tryptone soy broth (TSB, Solarbio), MS-222 (Sigma Aldrich), confocal bottom glass culture dish (20 mm in diameter, biosharp), 12 cm sterile glass culture dish (120 mm in diameter), and other conventional reagents such as analytical pure alcohol and xylene were all from Guoyao reagents.

[0024] 3. Instruments and equipment: Zebrafish independent single-frame circulating breeding system (Tecnplast); The fluorescence imaging analysis equipment was a laser confocal microscopy imaging system (SP8, Leica); Heating rod (purchased from Zhongshan New Force Electric Appliance Co., Ltd.).

[0025] Example 1 Application of Terminalia chebula as a feed additive in alleviating soybean meal-induced foodborne enteritis

[0026] 1. Experimental feed formula and preparation method with Terminalia chebula added in the zebrafish model

[0027] Table 1 Zebrafish soybean meal modeling and feed formula with Terminalia chebula added

[0028]

[0029] Note: 1. FM: fish meal group; 50SBM: soybean meal group; 0.5‰TBR: group with 0.5‰ Terminalia chebula added; 1‰TBR: group with 1‰ Terminalia chebula added; 2‰TBR: group with 2‰ Terminalia chebula added; In the vitamin additive in the formula, various vitamins refer to NRC, 1993.

[0030] (1) Pass fish meal, soybean meal, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix, etc. through a 60-mesh sieve. There are relatively large particles such as fish bones and soybean skins in fish meal and soybean meal. After pulverizing with a pulverizer, pass through a 60-mesh sieve. For vitamins and minerals with relatively low contents: VD3, VK3, VB12, thiamine, VB6, folic acid, copper sulfate, and sodium selenite need to be diluted 20 times to prepare the premix. Each kilogram of the mineral mixture contains: magnesium sulfate (MgSO4·2H2O) 60.530 g, ferrous sulfate (FeSO4·H2O) 23.110 g, copper sulfate (CuSO4·5H2O) 0.010 g, zinc sulfate (ZnSO4·H2O) 0.620 g, manganese sulfate (MnSO4·H2O) 1.640 g, potassium iodide (KI) 0.070 g, sodium selenite (NaSeO3) 0.005 g, and adjust to 1 kg with microcrystalline cellulose. Each kilogram of the above vitamin premix contains: vitamin B1 (Thiamin) 0.05 g, vitamin B2 (Riboflavin) 0.55 g, vitamin B6 (pyridoxine) 0.59 g, vitamin B12 (cyanocobalamine) 0.83 g, pantothenic acid 2.89 g, folic acid 0.40 g, inositol 19.39 g, niacin 2.24 g, biotin 4.91 g, vitamin C (ascorbic) 7.16 g, vitamin A (vitamin A) 2.40 g, vitamin D (vitamin D) 0.40 g, vitamin E (vitamin E) 12.55 g, vitamin K (Vitamin K) 0.80 g, and adjust to 1 kg with microcrystalline cellulose.

[0031] (2) Weigh fish meal, soybean meal, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix, and Terminalia chebula Retz. drug respectively according to the requirements of the weight percentages of each component in the feed formula.

[0032] (3) Stir and mix the weighed fish meal, soybean meal, and wheat flour evenly.

[0033] (4) After fully stirring and mixing microcrystalline cellulose, mineral premix, vitamin premix, and Terminalia chebula Retz. evenly, add corn starch and mix evenly. Add the evenly mixed mixture to the mixture in step (3) and continue to stir until evenly mixed.

[0034] (5) Weigh fish oil according to the formula table, add it to the mixture in step (4), rub and stir the large oil droplets fully until the fish oil is evenly distributed in the mixture.

[0035] (6) Pass the above mixture through a 60-mesh sieve. Crush the larger particles with a pulverizer and then pass them through the 60-mesh sieve until no particles remain.

[0036] (7) Add a certain proportion of water to the mixture in step (6) and stir it to make the mixture into a state where it can be formed into a ball by hand and dispersed when put down.

[0037] (8) Use a twin-screw granulator with a diameter of 2 mm. Control the extrusion pressure, corresponding rotation speed and temperature. After preheating, slowly add the mixture in step (7) into the granulator. Place the granulated feed in a pre-prepared dryer for drying. Control the temperature of the dryer at 55 - 60 °C and dry the moisture to about 10%.

[0038] (9) Crush the dried feed, pass it through a 60-mesh sample sieve to obtain the corresponding powdered feed, and store it in a -20 °C refrigerator in separate tubes for later use.

[0039] 2. Preparation of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) fry

[0040] (1) One week in advance, feed the fluorescently labeled zebrafish Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) that are separately cultured for males and females until they are full. Control the breeding water temperature at 28 ± 0.5 °C, the light time at 14 h, and the dark time at 10 h. The environment of the zebrafish breeding system is pH 7.0 - 8.0, salinity 0.25 - 0.50 ‰, dissolved oxygen 5 - 8 mg / L, total ammonia nitrogen less than 0.02 mg / L. For other conditions, refer to the requirements provided by the National Zebrafish Breeding Center (http: / / www.zfish.cn).

[0041] (2) Separate one male and one female adult fish of the Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) fluorescent strains with a mating fish tank overnight. Remove the partition the next morning and let them naturally chase and spawn for one hour. Collect the fish eggs and divide them into 12 cm × 5 cm glass culture dishes, about 100 eggs per dish, to obtain embryos with double fluorescent labels of Tg(lyz:DsRED2); Tg(mpeg1:EGFP). The stock solution formula of 30× Danieau’s buffer used for cultivating zebrafish larvae is as follows:

[0042] Table 2 Stock solution of 30× Danieau’s buffer

[0043]

[0044] (3) Incubate in a light incubator (28 ± 1°C, 14 h of light, 10 h of darkness), change water daily and pick out dead white fish eggs. By the 5th day of cultivation (5 dpf), prepare for the sub-packaging of fry and start the subsequent modeling experiments.

[0045] 3. Feeding protocol for the SBMIE modeling of zebrafish larvae

[0046] (1) After the double transgenic zebrafish Tg(lyz:DsRED2); Tg(mpeg1:EGFP) are hatched until the 5th day (5 pdf), sub-pack them into sterile round Petri dishes with a diameter of 150 mm, 30 tails per dish. The experiment is divided into five groups, namely: Group 1: FM, Group 2: 50SBM, Group 3: 0.5‰ TBR, Group 4: 1‰ TBR, Group 5: 2‰ TBR. Each group has three biological replicates.

[0047] (2) Feed each group with the corresponding feed. First, dissolve the feed in 0.3× Danieau’s buffer before feeding. Feed twice a day, and the feeding time period is: 9:00 - 9:30, 16:00 - 16:30. After feeding for half an hour, change to 0.3× Danieau’s buffer culture solution.

[0048] (3) After the feeding is completed on the 9th day (9 dpf), replace all the 0.3× Danieau’s buffer to keep the Petri dish clean and tidy for the subsequent in vivo imaging analysis of fry.

[0049] 4. In vivo imaging method for enteritis and remission degree of zebrafish larvae

[0050] (1) Prepare 25×100 mL of MS-222 stock solution (400 mg of Tricaine powder, 97.9 ml of deionized water, 2.1 ml of 1M Tris-HCL PH = 9.0) with sterile water and store it at 4°C. Dilute its working solution to 1× MS-222 working solution with deionized water; prepare 1% (w / v) low melting point agarose with deionized water and let it stand at room temperature for fixing the fry for imaging.

[0051] (2) Anesthesia: Inhale the corresponding group of juvenile fish into 1× MS-222 working solution and anesthetize for about 30 s until a large number of fry are in an anesthetized state.

[0052] (3) Fixation: Heat the 1% low melting point agarose prepared in (1) with a microwave oven until it dissolves and keep its temperature at 29°C. Add the anesthetized fry in (2) into a confocal Petri dish with a diameter of 20 mm, add the corresponding low melting point agarose solution, quickly adjust the posture of the fry so that the enlarged part of the foregut and the mid- and hindgut are not covered by the yolk, and keep the fry lying on its side at the bottom of the confocal Petri dish.

[0053] (4) Imaging: As soon as possible within 2 hours, take frontal and lateral photos of all the fixed fry in (3) under a laser confocal microscope (SP8, Leica). Set the software and simultaneously complete imaging in three channels: red fluorescence (excitation light 638 nm), green fluorescence (excitation light 488 nm), and white light under a 10× objective lens.

[0054] (5) Statistical analysis: After data collection, use Leica Application Suite X and ImageJ software to process the image data and count the number of fluorescent cells (cell size is approximately 10 microns) in the mid and hindgut. Perform statistical analysis using GraphPad Prism 7.0.

[0055] In this example, first, by hybridizing two immune cell markers, Tg(lyz:DsRED2) and Tg(mpeg1:EGFP), fry of Tg(lyz:DsRED2);Tg(mpeg1:EGFP) with both red and green fluorescent labels can be obtained, enabling the evaluation of two very important immune cells involved in innate immunity, neutrophils (red) and macrophages (green), in a single strain. By formulating and feeding powdered feed, a foodborne enteritis model can be rapidly established at the juvenile fish level to study the impact on acute inflammatory responses, and the effect of an ingredient that can alleviate the corresponding foodborne enteritis, Terminalia chebula, can be evaluated based on soybean meal feed.

[0056] According to the modeling strategy at the innate immunity stage, on the 9th day (9 dpf), as Figure 1 shown, fluorescently labeled neutrophils and macrophages appear in the mid and hindgut regions of the FM group, SBM group, and different concentrations of Terminalia chebula groups. The number of fluorescently labeled cells in the mid and hindgut regions of the FM group, SBM group, and different concentrations of Terminalia chebula groups was counted. Compared with the FM group, it was found that there was a greater aggregation of fluorescently labeled neutrophils and macrophages in the SBM group, indicating that feeding soybean meal feed can cause acute inflammatory responses. Compared with the SBM group, after adding 0.5‰ TBR, 1‰ TBR, and 2‰ TBR to the soybean meal feed, the aggregation of neutrophils and macrophages in the mid and hindgut can be alleviated to a certain extent. Among them, adding 1‰ TBR to the soybean meal feed has the best effect on alleviating neutrophil aggregation, and adding 1‰ TBR to the soybean meal feed has the best effect on alleviating macrophage aggregation.

[0057] The results of this experiment show that in the soybean meal-induced 9 dpf zebrafish enteritis model, in the group with 1‰ TBR added to the soybean meal feed, the reduction effect on the aggregation of neutrophils and macrophages in the mid and hindgut is the best, indicating that adding Terminalia chebula to the feed can alleviate the aggregation of neutrophils and macrophages in the mid and hindgut and can relieve zebrafish intestinal inflammation.

[0058] Example 2: Effect of Terminalia chebula on the composition of intestinal flora in zebrafish SBMIE model

[0059] Wild-type zebrafish with similar numbers and body sizes were divided into three tanks and fed twice a day with 1‰ TBR, FM, and SBM respectively according to the method described in Example 1. After stopping water supply and feeding for half an hour at 8:00 am and 5:00 pm every day in the circulating water system, the circulating water system was then turned on. After two weeks of feeding, their intestines were taken for 16S rRNA gene sequencing.

[0060] The results are as Figure 2 shown. At the phylum level, compared with SBM, in TRB: Proteobacteria has a large number of pathogenic bacteria, such as Escherichia coli, Escherichia, Salmonella, Vibrio, etc. The decrease in Proteobacteria indicates a significant reduction in the number of pathogenic bacteria in the intestine, which is beneficial to the homeostasis of the body. The increase in Bacteroidetes means an increase in the body's metabolic level, indicating that Terminalia chebula has a good effect of inhibiting the reproduction of harmful bacteria and promoting the reproduction of probiotics.

[0061] The results are as Figure 3 shown. At the genus level, compared with SBM, in TRB: The increase in Bacteroides can promote the degradation of polysaccharides and help release energy from dietary fiber and starch. The decrease in the pathogenic genus Brevibacillus also indicates that Terminalia chebula has the effect of inhibiting pathogenic genera. Some studies have shown that ZOR0006 belongs to the family Erysipelotrichaceae, which is related to host metabolic disorders and inflammatory diseases and has the ability to metabolize plant polysaccharides. The significant decrease in ZOR0006 in the Terminalia chebula group indicates a change in its inflammatory metabolism. Bifidobacterium is widely regarded as a member of probiotics, which has functions such as anti-inflammatory, improving blood sugar levels, reducing blood lipid levels, and enhancing the body's immunity. The increase in Faecalibacterium, an important producer of butyric acid, has an anti-inflammatory effect. At the genus level, it also shows that Terminalia chebula has a good effect of inhibiting the reproduction of harmful bacteria and promoting the reproduction of probiotics.

[0062] The results are as Figure 4 shown. The Shannon-Wiener index curve shows that the species richness and diversity of the Terminalia chebula group are higher than those of the soybean meal group, indicating that the intestinal stability of the Terminalia chebula group is higher.

[0063] Example 3: Effect of Terminalia chebula on inhibiting Aeromonas hydrophila in vitro

[0064] 1. Activation of the strain: Take out the frozen bacterial liquid from the -80°C refrigerator. After it melts, mix it evenly. Add 10 ml of LB liquid medium to a test tube, and inoculate 100 μL of the bacterial liquid into it with a pipette gun. Then place it in a constant temperature shaker at 37°C and culture it overnight at 800 rpm until the medium in the test tube becomes turbid.

[0065] 2. Cultivation of single colonies: Prepare LB solid medium and sterilize it in a high-temperature and high-pressure sterilizer. After it cools to an appropriate temperature, pour the plate. Dip the bacterial liquid with an inoculation needle and use the three-zone streaking method to culture single colonies. Culture it overnight in a constant temperature incubator at 37°C until single colonies grow.

[0066] 3. Inoculation of single colonies: Prepare a test tube containing 10 mL of LB liquid medium. Use sterilized forceps to pick up a small pipette tip, pick up a single colony with the front end, and then vertically place it into the test tube. Culture it overnight in a constant temperature shaker at 37°C and 800 rpm until the medium in the test tube becomes turbid.

[0067] 4. Preparation of aqueous extract of Terminalia chebula Retz.: Take 5 g of the dry and mature fruits of Terminalia chebula Retz., soak them in 100 mL of distilled water for 24 h, then boil them for 30 min. Filter them with four layers of gauze, collect the drug residue and repeat the above operation twice. Centrifuge the collected liquid at 2000 rpm for 10 min, take the supernatant, boil and concentrate all the supernatant to 25 ml. Sterilize it in a high-pressure steam sterilizer and then place it in a 4°C refrigerator for standby.

[0068] 5. Preparation of drug sensitivity test strips: Take drug sensitivity test strips in a sterile operating table and soak them in the original solution of Terminalia chebula Retz. (concentration 200 mg / ml) for 24 h. Take them out and place them in a petri dish, then dry them in an oven.

[0069] 6. Take 100 μl (10 7 CFU / ml) of Aeromonas hydrophila and spread it on a TSB plate. Place the drug sensitivity test strip on the TSB plate. After culturing at 29°C for 12 h, use a ruler to measure the size of the inhibition zone.

[0070] The results are as Figure 5 : The aqueous extract of Terminalia chebula Retz. at 200 mg / ml has an obvious antibacterial effect on Aeromonas hydrophila. The diameter of the inhibition zone of Terminalia chebula Retz. against Aeromonas hydrophila is 12.3 mm (the diameter of the filter paper is 6 mm).

[0071] Example 4 Determination of the minimum inhibitory concentration and minimum bactericidal concentration of Terminalia chebula Retz. against Aeromonas hydrophila

[0072] 1. Inoculate in a sterile operating table according to the volume ratio of Aeromonas hydrophila:TSB liquid medium = 1:1000, and culture it in a constant temperature incubator at 29°C for 12 h. Use the ten-fold serial dilution plating method to measure the bacterial concentration and dilute it to 10 7 CFU / ml with the corresponding medium.

[0073] 2. Add 10 μl of Aeromonas hydrophila bacterial solution to 1 ml of TSB medium containing Terminalia chebula aqueous extract at different concentrations. The concentrations of Terminalia chebula aqueous extract are 100 mg / ml, 50 mg / ml, 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, and 3.125 mg / ml respectively. Observe the results after culturing at 29 °C and 180 rpm for 12 h.

[0074] 3. The minimum inhibitory concentration is the lowest concentration at which the turbidity of the culture solution is not visible to the naked eye. The samples with no visible turbidity to the naked eye are further streaked on a plate and cultured at the corresponding temperature for 12 h. The lowest concentration without bacterial growth is the minimum bactericidal concentration.

[0075] The minimum inhibitory concentration of Terminalia chebula against Aeromonas hydrophila is 2.5 mg / ml, and the minimum bactericidal concentration is 12.5 mg / ml.

[0076] Example 5: Effect of Terminalia chebula on the growth of Aeromonas hydrophila

[0077] Set up a blank control group and a drug group. Inoculate 10 7 CFU / ml of Aeromonas hydrophila into TSB medium supplemented with Terminalia chebula aqueous extract (final concentration 0.75 mg / ml) at a volume ratio of 1 / 100 in a sterile manner, and culture at 29 °C and 180 rpm. Take the bacterial solution every 1 h after 4 h of culture, and measure the absorbance at a wavelength of 600 nm.

[0078] As Figure 6 shown, 0.75 mg / ml of Terminalia chebula aqueous extract has an obvious inhibitory effect on Aeromonas hydrophila.

[0079] Example 6: Effect on the survival rate of each group after Aeromonas hydrophila challenge

[0080] First, to determine the LD50 (median lethal concentration), three-month-old adult zebrafish are fed with fish meal according to 2% of their body weight for one week of acclimation. Set up a challenge experiment with Aeromonas hydrophila at concentration gradients of 1×10 5 CFU / ml, 1.5×10 5 CFU / ml, 2×10 5 CFU / ml, 2.5×10 5 CFU / ml, and 3×10 5 CFU / ml. Measure the median lethal concentration. Set up three parallel fish tanks (length × width × height: 27 cm × 16 cm × 13 cm) for each concentration, with a heating rod inside to maintain the water temperature at 28 °C. Put 10 adult zebrafish in each parallel tank. Record the death situation of zebrafish after 10 days of challenge. The final test results show that the median lethal concentration of Aeromonas hydrophila for zebrafish is 1×10 5 CFU / ml.

[0081] Then, an Aeromonas hydrophila challenge test on zebrafish was conducted. According to the method described in Example 1, the FM group, SBM group, and TBR group (50% soybean meal replacing fish meal feed supplemented with 1‰ Terminalia chebula) were set up. After adult zebrafish at three months of age were fed fish meal for one week according to 2% of the average body weight of the fish body, they were continuously fed the feeds of each group for six weeks. Three parallel fish tanks (length × width × height: 27 cm × 16 cm × 13 cm) were set up, and a heating rod was placed inside to maintain the water temperature at 28°C. 10 zebrafish were placed in each parallel, and Aeromonas hydrophila bacterial solution was added to each to make the final concentration reach 1×10 5 CFU / ml. One-third of the water was changed every morning and the bacterial solution concentration was replenished, and the death situations of each group were observed and recorded. As Figure 7 shown, the 10-day survival rates of the FM group, SBM group, and TBR group were 70%, 43%, and 70% respectively. The survival rate of the TBR group was significantly increased compared with the SBM group, and the survival rate returned to the normal level compared with the FM group. The results showed that Terminalia chebula could effectively inhibit the infection of Aeromonas hydrophila and effectively improve the survival rate level of zebrafish.

Claims

1. Application of Terminalia chebula Retz. in preparing drugs for relieving foodborne intestinal inflammation in fish.

2. Application of Terminalia chebula Retz. in preparing products for improving intestinal flora of fish with foodborne intestinal inflammation.

3. The application according to claim 2, characterized in that, Enhance the abundances of Faecalibacterium, Bifidobacterium, and Bacteroides.

4. Application of Terminalia chebula Retz. in preparing drugs for inhibiting Aeromonas hydrophila infection in fish.

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