Application of solidago decurrens in relieving soybean meal induced enteritis, regulating intestinal flora and inhibiting bacteria

Abstract

The invention discloses application of solidago decurrens in relieving fish food-borne intestinal inflammation, regulating intestinal flora and inhibiting bacteria, based on a 9-day soybean meal induced zebrafish enteritis model, 0.5-0.2% o of solidago decurrens dry whole herb is added into feed, so that aggregation of neutrophils and macrophages in middle and rear intestines can be relieved, the food-borne intestinal inflammation of zebrafish can be relieved, and the intestinal flora can be regulated. 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 0.05% o of the dried solidago decurrens herb into the feed; an aeromonas hydrophila in-vitro bacteriostasis experiment shows that the solidago decurrens aqueous extract has a remarkable bacteriostasis effect on the aeromonas hydrophila, and an aeromonas hydrophila in-vivo bacteriostasis experiment shows that the bean pulp feed added with 0.05% o of dried solidago decurrens whole herb can remarkably improve the survival rate of zebra fish infected by the aeromonas hydrophila.

Description

Technical Field

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

[0002] The growing world population is driving the demand for more readily available, nutritious aquatic products. According to the "State of World Fisheries and Aquaculture 2022" report released by the Food and Agriculture Organization of the United Nations (FAO), global aquatic animal production reached 178 million tons in 2020, of which aquaculture accounted for 49%. China leads the world in annual aquaculture production. From 1990 to 2020, global aquaculture production increased by 609%, with an average annual growth rate of 6.7%.

[0003] Aquaculture production is increasing year by year, and feeding costs will also rise accordingly. For economic benefits, replacing fish meal with plant-based protein is an optional strategy. Soybean meal is an important choice of plant protein source. The anti-nutritional factors in it can induce intestinal inflammation. [1] Feed additives have a certain application in soybean meal replacement feed. Traditional Mongolian and Tibetan medicine sea buckthorn, natural substances sinomenine, and gallic acid can alleviate soybean meal-induced enterohepatitis. [2-4] .

[0004] The annual increase in aquaculture production has led to an increase in the harm caused by aquaculture pathogens. Aeromonas hydrophila is a Gram-negative bacterium that is widely distributed in nature and is a zoonotic bacterium that can cause disease in a wide range of hosts, including mammals, amphibians, reptiles, and fish. Aeromonas hydrophila can cause bacterial septicemia in many fish species. Infected fish suffer from surface bleeding, congestion, and visceral swelling, with 80% to 100% mortality within 1 to 2 weeks. The extremely high mortality rate has caused huge economic losses to the aquaculture industry. [5-10] .

[0005] Antibiotics are the main means of defense against pathogens in aquaculture, but with the abuse of antibiotics, many drug-resistant bacteria have been selected. The international ban on antibiotics has greatly promoted the development of new antibiotic substitutes. Natural medicinal plant resources have many applications in the prevention and control of aquaculture pathogens, and oregano

[11] , Astragalus

[12] , Dendrobium officinale

[12] Natural medicinal substances such as licorice have certain antibacterial properties, and licorice can reduce the mortality rate of rainbow trout infected with Yersinia ruckeri.

[13] .

[0006] Solidago sphenanthera, belonging to the genus Solidago of the Asteraceae family, has the functions of dispelling wind and heat, detoxifying and reducing swelling.

[14] It is mainly used for treating wind-heat cold, headache, sore throat, lung-heat cough, etc. In addition, it can treat upper respiratory tract infection, tinea manus et pedis, herpes zoster, oral ulcer, hypotension and other diseases. [15,16] In terms of antibacterial, the decoction of Solidago virgaurea has a strong inhibitory effect on Staphylococcus aureus, and its antibacterial effect on Gram-positive bacteria is stronger than that on Gram-negative bacteria.

[17] In terms of anti-inflammatory, the gargle of Solidago virgaurea can significantly reduce oral mucositis in fasting patients after general anesthesia.

[18] Combined with other Chinese herbal medicines, it can treat transmissible gastroenteritis of pigs, infectious laryngotracheitis of chickens, mastitis of dairy cows, etc. In terms of antiviral, Solidago virgaurea has anti-AIDS virus activity.

[0007] There are also reports on the treatment of acute tonsillitis and mycotic vaginitis with Solidago virgaurea, but there is a lack of research on fish foodborne intestinal inflammation. The inhibitory effects of Solidago virgaurea on Staphylococcus aureus, Staphylococcus faecalis, Pseudomonas aeruginosa, Micrococcus luteus, Morganella and other bacteria

[19] have been reported, but there is no report on its resistance to the aquatic pathogenic bacterium Aeromonas hydrophila.

[20] Summary of the Invention

[0008] The purpose of the present invention is to provide new uses of Solidago virgaurea, including its application in the preparation of drugs for inhibiting the infection of Aeromonas hydrophila in fish, its application in the preparation of drugs for alleviating fish foodborne intestinal inflammation, and its application in the preparation of products for improving the intestinal flora of fish with foodborne intestinal inflammation.

[0009] In order to achieve the above purpose, the present invention adopts the following technical measures:

[0010] Application of Solidago virgaurea in the preparation of drugs for alleviating fish foodborne intestinal inflammation: In the specific embodiment of the present invention, a foodborne enteritis model was established by feeding zebrafish larvae with a feed in which 50% of the protein source was replaced by soybean meal. Solidago virgaurea was added to the feed in which 50% of the protein source was replaced by soybean meal at 0.5‰, 1‰, and 2‰ respectively. The innate immune cell imaging analysis of 9dpf zebrafish larvae was carried out to test the improvement effect of inflammation. The results showed that compared with the feed group in which 50% of the protein source was replaced by soybean meal, adding Solidago virgaurea to the soybean meal feed could alleviate the aggregation of neutrophils and macrophages in the mid and hindgut to a certain extent. And adding 2‰ Solidago virgaurea had the best effect on alleviating neutrophil aggregation, and adding 1‰ Solidago virgaurea had the best effect on alleviating macrophage aggregation. Therefore, Solidago virgaurea has a relieving effect on fish foodborne intestinal inflammation.

[0011] Application of Solidago virgaurea in preparing products for improving intestinal flora of fish with foodborne intestinal inflammation: In the specific embodiments of the present invention, after feeding 3-month-old zebrafish with a protein source feed, a feed with 50% of the protein source replaced by soybean meal, and a 50% soybean meal replacing protein source feed containing 0.5‰ Solidago virgaurea for 2 weeks, the whole intestine of each group was randomly sampled for 16S rRNA gene sequencing to determine the regulatory effect of Solidago virgaurea on the intestinal flora. The results showed that adding Solidago virgaurea could regulate the intestinal flora of fish with foodborne intestinal inflammation. At the genus level, the abundances of Bacteroides, Parabacteroides, Cetobacterium, and Akkermansia were increased, improving the intestinal homeostasis of fish with foodborne intestinal inflammation.

[0012] Application of Solidago virgaurea in preparing drugs for inhibiting Aeromonas hydrophila infection in fish: In the specific embodiments of the present invention, the antibacterial effect of the water extract of Solidago virgaurea on Aeromonas hydrophila was preliminarily verified by measuring the inhibition zone of Solidago virgaurea against Aeromonas hydrophila. The minimum inhibitory concentration of Solidago virgaurea against Aeromonas hydrophila was 50 mg / ml, and the minimum bactericidal concentration was 150 mg / ml. Further, after feeding 3-month-old zebrafish with 0.5‰ Solidago virgaurea for 6 weeks, a 10-day Aeromonas hydrophila challenge experiment was conducted on the zebrafish to determine the in vivo anti-Aeromonas hydrophila ability of Solidago virgaurea in adult zebrafish. The results showed that 0.5‰ Solidago virgaurea could significantly improve the survival rate of zebrafish under Aeromonas hydrophila infection.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] The present invention first proposes the application of Solidago virgaurea in alleviating foodborne enteritis of zebrafish caused by soybean meal, which can alleviate enteritis in juvenile zebrafish while reducing feed costs, providing a feasible reference for feed additives for economic fish.

[0015] The present invention first proposes the application of Solidago virgaurea in regulating the intestinal flora of zebrafish. After feeding adult zebrafish with a soybean meal feed containing 0.5‰ Solidago virgaurea instead of a fish meal feed for two weeks, the abundances of some beneficial bacteria were increased and the abundances of some harmful bacteria were decreased, providing feasibility for regulating the intestinal flora of economic fish.

[0016] The present invention first proposes the growth inhibitory effect of Solidago virgaurea on Aeromonas hydrophila, which is harmful to fish. Solidago virgaurea can be used as a new type of antibiotic alternative feed additive. In addition, the Solidago virgaurea used is a natural medicinal plant. As a feed functional additive derived from Chinese herbal medicine, it has the advantages of environmental protection, non-toxicity, high cost-effectiveness, and easy handling. Description of the Drawings

[0017] Figure 1 It is a fluorescence imaging result diagram of intestinal neutrophils and macrophages of juvenile zebrafish (9 dpf) after adding Solidago virgaurea to the soybean meal feed.

[0018] Figure 2 Heatmap of the relative abundances at the phylum level after 16S rRNA gene sequencing of the intestine of 3-month-old zebrafish after adding Solidago virgaurea to soybean meal feed. The abscissa FM represents the fish meal group, SBM represents 50% soybean meal replacing fish meal, and SDL represents the 50% soybean meal replacing fish meal group with 0.5‰ Solidago virgaurea added.

[0019] Figure 3 Heatmap of the relative abundances at the genus level after 16S rRNA gene sequencing of the intestine of 3-month-old zebrafish after adding Solidago virgaurea to soybean meal feed. The abscissa FM represents the fish meal group, SBM represents 50% soybean meal replacing fish meal, and SDL represents the 50% soybean meal replacing fish meal group with 0.5‰ Solidago virgaurea added.

[0020] Figure 4 Inhibitory zone diagram of the water extract of Solidago virgaurea against Aeromonas hydrophila.

[0021] Figure 5 Growth curve of the co-culture of the water extract of Solidago virgaurea and Aeromonas hydrophila.

[0022] Figure 6 Survival rate diagram after 10 days of challenge with Aeromonas hydrophila after feeding adult zebrafish for six weeks with 0.5‰ Solidago virgaurea added to soybean meal feed. Detailed implementation methods

[0023] 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 of 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.

[0024] The following are the experimental materials, reagents, and instrument equipment involved in the embodiments:

[0025] 1. Biological materials: The AB wild-type zebrafish (Danio rerio) used in this experiment, transgenic zebrafish lines, 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 hybridizing two single-color fluorescent lines. The strains used in this experiment were provided by the National Aquatic Germplasm Resource Bank - Sub-bank of Characteristic Aquatic Animal Germplasm Resources. Among them, the Aeromonas hydrophila was coded as Aer-028 and was isolated from the liver of grass carp in 2008.

[0026] 2. Reagents and consumables: Whole dried Solidago virgaurea (purchased from Shandu), tryptone soy broth (TSB, Solarbio), low melting point agarose (UltraPureTM LMP Agarose, invitrogen), MS-222 (SigmaAldrich), 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 absolute ethanol are all from Sinopharm Chemical Reagent Co., Ltd.

[0027] 3. Instruments and equipment: Zebra fish independent single-frame circulating breeding system (Tecnplast); the fluorescence imaging analysis equipment is a laser confocal microscopy imaging system (SP8, Leica); heating rod (purchased from Zhongshan Xinshili Electric Appliance Co., Ltd.).

[0028] Example 1 Application of Solidago virgaurea as a feed additive in alleviating soybean meal-induced enteritis in zebrafish

[0029] 1. Experimental feed formula and preparation method of adding Solidago virgaurea in zebrafish model

[0030] Table 1 Zebrafish soybean meal modeling and Solidago virgaurea-added feed formula

[0031]

[0032]

[0033] Note: FM: fish meal group; 50SBM: soybean meal group; 0.5‰SDL: group added with 0.5‰ Solidago virgaurea; 1‰SDL: group added with 1‰ Solidago virgaurea; 2‰SDL: group added with 2‰ Solidago virgaurea; for various vitamins in the vitamin additive in the formula, refer to NRC, 1993.

[0034] (1) Pass fish meal, soybean meal, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix, etc. through a 60-mesh sieve. The larger particles in fish meal and soybean meal are pulverized with a pulverizer and then passed through a 60-mesh sieve;

[0035] (2) Weigh fish meal, soybean meal, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix, and Solidago virgaurea according to the weight percentages of each component in Table 1;

[0036] (3) Stir and mix the weighed fish meal, soybean meal, and wheat flour thoroughly;

[0037] (4) After thoroughly stirring and mixing microcrystalline cellulose, mineral premix, vitamin premix, and Solidago virgaurea, add corn starch and mix evenly. Then mix the above mixture into (3) and stir until evenly mixed;

[0038] (5) Weigh the fish oil according to Table 1, add it to (4), rub and stir the large oil droplets until the fish oil is evenly distributed in the mixture;

[0039] (6) Pass the above mixture through a 60-mesh sieve, crush the larger particles with a pulverizer and then pass them through a 60-mesh sieve until no particles remain;

[0040] (7) Add a certain proportion of water to the mixture in step (6) and stir it to make the mixture in a state where it can be kneaded into a ball by hand and dispersed when put down;

[0041] (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 (7) to the granulator, and place the granulated feed in a pre-prepared dryer for drying. The temperature of the dryer is controlled at 55 - 60 °C, and the moisture is dried to about 10%;

[0042] (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.

[0043] 2. Preparation of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) fry

[0044] (1) One week in advance, feed the transgenic zebrafish Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) separately raised by gender until they are full. Control the breeding water temperature at 28 ± 0.5 °C, the light time is 14 h, the darkness is 10 h, the environment of the zebrafish breeding system is pH 7.0 - 8.0, the salinity is 0.25 - 0.50 ‰, the dissolved oxygen is 5 - 8 mg / L, the total ammonia nitrogen is less than 0.02 mg / L, and other conditions refer to the requirements provided by the National Zebrafish Breeding Center (http: / / www.zfish.cn);

[0045] (2) Separate an adult male and female 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 lay eggs for 1 h. Collect the fish eggs and divide them into glass culture dishes to obtain embryos with double fluorescence labeling of Tg(lyz:DsRED2); Tg(mpeg1:EGFP). The stock solution formula of 30×Danieau’s buffer used for cultivating zebrafish fry is as follows:

[0046] Table 2 Stock solution of 30×Danieau’s buffer

[0047]

[0048] (3) Incubate in a light incubator (28 ± 1°C, 14 h of light and 10 h of darkness), change water daily and pick out dead eggs. On the 5th day after fertilization (5 dpf), separate the fry and start the subsequent modeling experiments.

[0049] 3. Feeding protocol for zebrafish larvae SBMIE modeling

[0050] (1) After culturing the double-fluorescent zebrafish Tg(lyz:DsRED2); Tg(mpeg1:EGFP) until the 5th day after fertilization (5 pdf), transfer them into sterile round culture dishes with a diameter of 150 mm, 20 tails per dish. The experiment is divided into five groups, namely: Group 1: FM, Group 2: 50SBM, Group 3: 0.5‰ SDL, Group 4: 1‰ SDL, Group 5: 2‰ SDL. Each group has three biological replicates.

[0051] (2) Feed each group with the corresponding feed. Dissolve the feed in Danieau’s buffer with a final concentration of 0.3×, and feed twice a day. The feeding time periods are: 9:00 - 9:30, 16:30 - 17:30. Replace the culture solution with 0.3× Danieau’s buffer half an hour after feeding.

[0052] (3) After feeding on the 9th day after fertilization (9 dpf), replace all the 0.3× Danieau’s buffer to keep the culture dish clean and tidy for subsequent in vivo imaging analysis of the fry.

[0053] 4. In vivo imaging method for enteritis and remission degree of zebrafish larvae

[0054] (1) Prepare a 25×100 mL stock solution of MS-222 with sterile water (400 mg of Tricaine powder, 97.9 mL of sterilized water, 2.1 mL of 1M Tris-HCl with pH = 9.0), store it at 4°C, and dilute its working solution to 1× with dd H2O; prepare 0.5% low melting point agarose with dd H2O and let it stand at room temperature for fixing the fry for imaging.

[0055] (2) Aspirate the corresponding group of juvenile fish into 1× MS-222 to anesthetize a large number of fry.

[0056] (3) Heat the 0.5% low melting point agarose prepared in (1) with a microwave oven until it dissolves. When its temperature is maintained at 29°C, put the anesthetized fry in (2) into the 0.5% low melting point agarose solution, and quickly adjust the posture of the fry so that the fry lies on its side at the bottom of the confocal culture dish.

[0057] (4) Take pictures of the fixed fry in (3) under a laser confocal microscope (SP8, Leica), set the software, and simultaneously complete the imaging of three channels: red fluorescence (excitation light 638 nm), green fluorescence (excitation light 488 nm), and white light under a 10× objective lens.

[0058] (5) After data acquisition, 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 μm) in the mid- and hindgut. Use GraphPad Prism 7.0 for statistical analysis.

[0059] In this example, by crossing two homozygous transgenic marker lines, Tg(lyz:DsRED2) and Tg(mpeg1:EGFP), fry were obtained that simultaneously contained neutrophils labeled with red fluorescence and macrophages labeled with green fluorescence, namely Tg(lyz:DsRED2);Tg(mpeg1:EGFP) fry. In one individual, two important immune cells (neutrophils, macrophages) involved in innate immunity can be observed simultaneously. The production and feeding of powdered feed enabled the observation of the effect of the foodborne enteritis model on the acute inflammatory response at the juvenile fish level, and the evaluation of the drug for relieving foodborne enteritis, Solidago virgaurea, based on soybean meal feed.

[0060] At 9 days post-fertilization (9 dpf), as Figure 1 shown, neutrophils and macrophages with corresponding fluorescence labels appeared in the mid- and hindgut regions of the FM group, SBM group, and different concentrations of Solidago virgaurea groups. The number of fluorescently labeled cells in the mid- and hindgut regions of the FM group, SBM group, and different concentrations of Solidago virgaurea groups was counted. Compared with the FM group, it was found that more neutrophils and macrophages labeled with fluorescence appeared in the SBM group, indicating that feeding soybean meal feed could cause an acute inflammatory response. Compared with the SBM group, after adding 0.5‰ SDL, 1‰ SDL, and 2‰ SDL to the soybean meal feed, the aggregation of neutrophils and macrophages in the mid- and hindgut could be alleviated to a certain extent. Among them, adding 2‰ SDL to the soybean meal feed had the best effect on alleviating neutrophil aggregation, and adding 1‰ SDL to the soybean meal feed had the best effect on alleviating macrophage aggregation.

[0061] In summary, in this example, by feeding different feeds during the innate immune stage and using imaging methods to establish a foodborne enteritis model, an acute inflammatory response in juvenile fish was induced. By adding Solidago virgaurea, the aggregation of neutrophils and macrophages in the mid- and hindgut was alleviated, and the acute inflammatory response in the mid- and hindgut was reduced, indicating that Solidago virgaurea has a relieving effect on foodborne intestinal inflammation in fish during the innate immune stage.

[0062] Application of Solidago virgaurea as a feed additive in regulating the intestinal flora of adult zebrafish

[0063] Three-month-old zebrafish were trained with fish meal for one week and divided into three groups: fish meal (FM), 50% soybean meal replacing fish meal (SBM), and 50% soybean meal replacing fish meal supplemented with 0.5‰ Solidago virgaurea (SDL), and fed for 2 weeks.

[0064] After the feeding experiment, the zebrafish were taken out, anesthetized with 1×MS-222, and after severing the tail and bleeding, the whole intestine of the zebrafish was taken out, quickly frozen in liquid nitrogen and stored at -80 °C for 16S rRNA gene sequencing of the flora.

[0065] As Figure 2 shown, at the phylum level, compared with SBM, the phylum Bdellovibrionota in the SDL group was significantly up-regulated (P<0.001). In addition, the phyla Bacteroidetes, Fusobacteria, and Verrucomicrobia all showed an increasing trend, while the phylum Proteobacteria showed a decreasing trend. Research has shown that Bdellovibrio can be used as an alternative to antibiotics and has a positive impact on the microbial community, which is beneficial to the healthy growth of Changfeng crucian carp. As Figure 3 shown, at the genus level, ZOR006 showed a downward trend. Some studies have shown that ZOR006 belongs to the family Erysipelotrichaceae of the phylum Firmicutes and is related to the metabolic disorders and inflammatory diseases of the host; the phylum Bacteroidetes can regulate the host immune homeostasis by producing short-chain fatty acids and enzymes in the processes of carbohydrate and protein metabolism. The genera Bacteroides and Parabacteroides under the phylum Bacteroidetes also showed an upward trend at the genus level; the phylum Fusobacteria can improve the host metabolism by producing short-chain fatty acids, vitamin B12, and bacteriocins. Cetobacterium is an important component of the phylum Fusobacteria, and it showed an increasing trend at the genus level compared with the SBM group; the phylum Verrucomicrobia mainly focuses on the study of the genus Akkermansia, which is related to lipid metabolism and can maintain intestinal homeostasis. Compared with SBM, the genus Akkermansia in the SDL group showed an upward trend; most of the phylum Proteobacteria are pathogenic bacteria, which can indicate the imbalance of the intestinal flora. The genus Acinetobacter belongs to the phylum Proteobacteria, and it also showed a downward trend at the genus level.

[0066] In summary, adding 0.5‰ Solidago virgaurea to the soybean meal feed can adjust the intestinal flora of three-month-old zebrafish by increasing the relative abundance of some beneficial bacteria and reducing the abundance of some harmful bacteria to a certain extent.

[0067] Example 3 In vitro antibacterial effect of Solidago virgaurea on Aeromonas hydrophila

[0068] The disc diffusion method was used to determine the size of the inhibition zone of Solidago virgaurea against Aeromonas hydrophila, and the steps were as follows:

[0069] 1. Grind the dried whole herb of Solidago virgaurea appropriately, weigh 5 g of the ground Solidago virgaurea, add 100 ml of dd H2O and soak for 24 h. After boiling the soaked Solidago virgaurea, keep it boiling for 30 min, filter with four layers of gauze. Add 100 ml of ddH2O to the filter residue, boil it and keep it boiling for 30 min, then filter with four layers of gauze. Collect the two filtrates, centrifuge at 2000 rpm for 10 min to remove the remaining residues, heat-concentrate the supernatant to 25 ml and then autoclave it to obtain a water extract of Solidago virgaurea at 200 mg / ml, and store it in a refrigerator at 4°C for later use.

[0070] 2. Aliquot part of the 200 mg / ml water extract of Solidago virgaurea, immerse blank filter paper discs for 24 h, transfer the filter paper discs to a petri dish, and take them out after 12 h in an oven at 65°C.

[0071] 3. Place the prepared filter paper discs containing the water extract of Solidago virgaurea on a TSB solid medium coated with Aeromonas hydrophila (10 7 CFU / ml). After culturing at 29°C for 12 h, measure the size of the inhibition zone with a ruler.

[0072] As Figure 4 shown, the inhibition zone of the 200 mg / ml water extract of Solidago virgaurea against Aeromonas hydrophila was 6.1 mm (the diameter of the blank filter paper disc was 6 mm), that is, at this concentration, Solidago virgaurea had a certain inhibitory effect on Aeromonas hydrophila.

[0073] Example 4 Determination of the minimum inhibitory concentration and minimum bactericidal concentration of Solidago virgaurea against Aeromonas hydrophila

[0074] The gradient dilution method was used to determine the minimum inhibitory concentration and minimum bactericidal concentration of Solidago virgaurea against Aeromonas hydrophila:

[0075] 1. Inoculate Aeromonas hydrophila aseptically into TSB medium and culture at 29°C for 12 h. Use the 10-fold dilution method to coat and measure the bacterial concentration respectively, and dilute it to 10 7 CFU / ml with the corresponding medium.

[0076] 2. Add 10 μl of the above-mentioned diluted Aeromonas hydrophila solution to 1 ml of medium containing water extracts of Solidago virgaurea at different concentrations. The concentrations of the water extracts of Solidago virgaurea were 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.

[0077] 3. The minimum inhibitory concentration is the lowest concentration at which turbidity of the culture medium is not visible to the naked eye. Samples with visible non-turbidity are further streaked on a plate and cultured at the corresponding temperature for 12 h. The lowest concentration at which no bacterial growth is observed is the minimum bactericidal concentration.

[0078] The minimum inhibitory concentration of Solidago virgaurea against Aeromonas hydrophila is 50 mg / ml, and the minimum bactericidal concentration is 150 mg / ml.

[0079] Example 5 Effect of Solidago virgaurea on the growth of Aeromonas hydrophila

[0080] 10 7 CFU / ml of Aeromonas hydrophila was aseptically inoculated into TSB medium containing 12.5 mg / ml of the aqueous extract of Solidago virgaurea at a volume ratio of 1 / 100 and cultured at 29 °C and 180 rpm. Bacterial liquid was taken every 1 h after 4 h of culture, and the absorbance at a wavelength of 600 nm was measured.

[0081] As Figure 5 shown, the aqueous extract of Solidago virgaurea at 12.5 mg / ml has a significant inhibitory effect on the growth of Aeromonas hydrophila.

[0082] Example 6 Inhibitory effect of Solidago virgaurea on Aeromonas hydrophila in vivo

[0083] After 3-month-old zebrafish were trained with fish meal for one week, they were continuously fed with fish meal (FM), 50% soybean meal replacing fish meal (SBM), and 50% soybean meal replacing fish meal feed containing 0.5‰ Solidago virgaurea (SDL) at 2% of the average body weight for 6 weeks. 30 zebrafish were randomly selected from each group and placed into 3 aerated water tanks (27 cm × 16 cm × 13 cm) containing 3 L of water, with 10 fish in each tank. The water temperature was maintained at 28 °C, and Aeromonas hydrophila was added to a final concentration of 1.0×10 5 CFU / ml. During the experiment, one-third of the water was changed every day and the corresponding bacterial liquid was added. The number of dead zebrafish was recorded every 24 h, and the observation lasted for 10 d in total. The results are as Figure 6 shown. The SDL group can improve the survival rate of zebrafish under Aeromonas hydrophila infection, and the survival rate (60%) after 10 d of virus challenge is higher than that of the SBM group (43.3%), but lower than that of the FM group (70.0%).

[0084] References:

[0085] [1] Zhang Wei, Tan Beiping, Pang Aobo, et al. Screening of potential biomarkers in the intestine of Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂ with soybean meal-induced enteritis [J]. Journal of Guangdong Ocean University, 2022, 42(4).

[0086] [2] LI M, ZHAO X, XIE J, et al. Dietary Inclusion of Seabuckthorn (Hippophae rhamnoides) Mitigates Foodborne Enteritis in Zebrafish Through the Gut-Liver Immune Axis[J]. Front Physiol, 2022, 13.

[0087] [3] XIE J Y, LI M, YE W D, et al. Sinomenine Hydrochloride Ameliorates Fish Foodborne Enteritis via α7nAchR-Mediated Anti-Inflammatory Effect Whilst Altering Microbiota Composition[J]. Front Immunol, 2021, 12.

[0088] [4] ZHAO X Y, ZHANG L, WU N, et al. Gallic acid acts as an anti-inflammatory agent via PPARγ-mediated immunomodulation and antioxidation in fish gut-liver axis[J]. Aquaculture, 2024, 578.

[0089] [5] HARIKRISHNAN R, BALASUNDARAM C. Modern Trends in Aeromonas hydrophila Disease Management with Fish[J]. Reviews in Fisheries Science, 2007, 13(4): 281 - 320.

[0090] [6] Wang S B, Xiong L W, Ji Z N. Bacteriostatic effect and mechanism of emodin on Aeromonas hydrophila[J]. Chinese Journal of Veterinary Medicine, 2023.

[0091] [7] DORICK J M, MACARISIN D, DUNN L, et al. Effect of aquaponic water and substratum material on biofilm formation by Aeromonas hydrophila[J]. Int J Food Microbiol, 2023, 404.

[0092] [8] GAO F, SHI X, ZHAO Y, et al. The role of CcPTGS2a in immune response against Aeromonas hydrophila infection in common carp (Cyprinus carpio)[J]. Fish Shellfish Immunol, 2023, 141: 109058.

[0093] [9] ZHANG M, YAN Q, MAO L, et al. KatG plays an important role in Aeromonas hydrophila survival in fish macrophages and escape for further infection[J]. Gene, 2018, 672: 156 - 64.

[0094]

[10] Sun Wen, Wang Yongjie, Bao Junjie, et al. Physiological damage of zebrafish intestine and intestinal flora dysbiosis caused by Aeromonas hydrophla[J]. Oceanologia et Limnologia Sinica, 2023, 54(4): 1191 - 8.

[0095]

[11] ALAGAWANY M, FARAG M R, SALAH A S, et al. The role of oregano herb and its derivatives as immunomodulators in fish[J]. Rev Aquacult, 2020, 12(4): 2481 - 92.

[0096]

[12] ANDLEEB S,TAHIR M,KHALID M,et al.Antibacterial and AntioxidantActivities of Traditional Herbs and Honey against Fish Associated BacterialPathogens[J].Pak J Zool,2014,46(4):933-40.

[0097]

[13] DARVISHI M,MEHRGAN M S,KHAJEHRAHIMI A E.Effect of Licorice(Glycyrrhiza glabra)Extract as an Immunostimulant on Serum and Skin MucusImmune Parameters,Transcriptomic Responses of Immune-Related Gene,and DiseaseResistance Against in Rainbow Trout(Oncorhynchus mykiss)[J].Front Vet Sci,2022,9.

[0098]

[14] Wang Yulan.Analysis of the Pharmacological Effects of Solidago virgaurea[J].Chinese Journal of Ethnomedicine and Ethnopharmacy,2010,19(6):150.

[0099]

[15] Qiu Mingyi,Li Xiaolan,Liu Supeng,et al.Effects of Solidago virgaurea on Some Indexes of the Cardiovascular System[J].Medical Information,2005,18(12):1730-1.

[0100]

[16] Liu Supeng,Qiu Mingyi,Wu Zhengping,et al.Effects of Solidago virgaurea on the Intestinal Smooth Muscle Movement of Animals[J].Lishizhen Medicine and Materia Medica Research,2006,17(11):2151-2.

[0101]

[17] Yang Jing,Zhang Weihua,Qiu Mingyi.Preliminary Study on the Bacteriostatic Effect in Vitro of Decoction and Extract of Solidago virgaurea[J].Journal of Sichuan of Traditional Chinese Medicine,2009,27(9):50-2.

[0102]

[18] Zhang Xiaoyun,Lu Jingbo,Dai Jinhua.Application of Solidago virgaurea Gargle in Preventing Oral Mucositis in Patients Fasted after General Anesthesia[J].Chinese Journal of Modern Nursing,2013,19(4):421-3.

[0103]

[19] Guo Yanrong, Zhang Yan, Song Guihua. Exploration of the Experience of Qingfei Jiedu Decoction plus Solidago virgaurea in the Treatment of Children with Lobarpneumonia [J]. Chinese Pediatrics of Integrated Traditional and Western Medicine, 2015, (4): 401 - 2.

[0104]

[20] FURSENCO C, CALALB T, UNCU L, et al. L.: A Review of Its Ethnomedicinal Uses, Phytochemistry, and Pharmacological Activities [J]. Biomolecules, 2020, 10(12).

Claims

1. Use of Solidago virgaurea in the preparation of a medicament for inhibiting Aeromonas hydrophila infection in fish.

2. Use of Solidago virgaurea in the preparation of a medicament for alleviating foodborne intestinal inflammation in fish.

3. Use of Solidago virgaurea in the preparation of a product for improving the intestinal flora of fish with foodborne intestinal inflammation.

4. The application according to claim 3, characterized in that Enhancing the abundances of the genera Bacteroides, Parabacteroides, Cetobacterium, and Akkermansia.

Images