Use of xanthohumol in the preparation of a medicament for the treatment of infections by aeromonas hydrophila in aquatic animals

Abstract

The application discloses application of xanthohumol in preparation of a medicine for resisting Aeromonas hydrophila infection of aquatic animals, and the xanthohumol can inhibit the activity of aerolysin of the Aeromonas hydrophila at a sub-inhibitory concentration, has a significant inhibitory effect on cell toxicity mediated by the aerolysin of the Aeromonas hydrophila, and further constructs an animal model infected by the Aeromonas hydrophila, and finds that the treatment of the xanthohumol can significantly improve the survival rate of the infected animals. The xanthohumol is one of main components of Chinese medicine hop, belongs to isoprenyl flavonoids, has biological functions such as bacteriostasis, anticancer, antioxidation, antiviral and hypoglycemic, and has high safety, and is an ideal medicine raw material or feed additive for preventing and treating the Aeromonas hydrophila infection of the aquatic animals.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to the application of xanthohumol in resisting Aeromonas hydrophila infection in aquatic animals. Background Technology

[0002] Xanthumol is a naturally derived isopentenyl flavonoid compound and a major chemical component of the traditional Chinese medicine hops. It possesses antibacterial, antiviral, anticancer, and antioxidant biological activities. Hops, also known as hops, has a bitter taste and slightly cooling properties; it has stomach-strengthening, digestive, sedative, diuretic, anti-tuberculosis, and anti-inflammatory effects, and is commonly used to treat indigestion, abdominal distension, tuberculosis, cough, and insomnia.

[0003] Aeromonas hydrophila is a common pathogen in freshwater aquaculture, causing disease in most farmed freshwater fish and also infecting crayfish, bullfrogs, and other species, posing a significant threat to the healthy development of the aquaculture industry. Furthermore, Aeromonas hydrophila is also a zoonotic pathogen, frequently causing diarrhea and septicemia in humans. Control of Aeromonas hydrophila in aquaculture primarily relies on antibiotics, but long-term irrational use has led to the emergence of drug-resistant bacteria, severely impacting treatment effectiveness. Therefore, there is an urgent need to develop new drugs to combat drug-resistant Aeromonas hydrophila infections in order to control fish diseases caused by these resistant bacteria.

[0004] The pathogenicity of *Aeromonas hydrophila* is closely related to the quantity and types of virulence factors it carries, among which aerolysins are the main virulence factors. Studies have found that *Aeromonas hydrophila* with aerolysin deficiency exhibits significantly reduced virulence both in vivo and in vitro. Therefore, aerolysins have become a potential target for developing anti-*Aeromonas hydrophila* infection treatments. Summary of the Invention

[0005] The purpose of this invention is to provide the application of xanthohumol in the preparation of drugs for treating Aeromonas hydrophila infection in aquatic animals. At concentrations that do not inhibit the growth of Aeromonas hydrophila, xanthohumol can directly inhibit the activity of aerolysins and has a significant inhibitory effect on aerolysin-mediated cytotoxicity, thereby improving the survival rate of the Aeromonas hydrophila infection model in crucian carp.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of xanthumol in the preparation of drugs against Aeromonas hydrophila infection in aquatic animals: Experiments verified that the minimum inhibitory concentration (MIC) of xanthumol against Aeromonas hydrophila is 128 μg / mL. At this experimental concentration, it had no effect on the growth of Aeromonas hydrophila, but it dose-dependently inhibited the hemolytic activity of Aeromonas hydrophila culture supernatant. This inhibition was statistically significant at xanthumol concentrations of 4 μg / mL and above. Furthermore, analysis of purified aerolysin protein revealed that xanthumol concentrations of 2 μg / mL and above significantly reduced the activity of purified aerolysin. These results indicate that xanthumol can directly inhibit aerolysin activity. Further experimental studies showed that xanthumol concentrations of 2 μg / mL and above significantly inhibited aerolysin-mediated cell damage in A549 cells, suggesting a protective effect against Aeromonas hydrophila infection. In addition, a *Aeromonas hydrophila* infection model was established in channel catfish. Results showed that oral administration of 20 mg / kg xanthohumol every 12 hours for 3 consecutive days significantly improved the survival rate of infected animals, reaching 65%, while the survival rate of the positive control group was only 10%. These studies indicate that xanthohumol can be used as a veterinary drug to treat *Aeromonas hydrophila* infection in aquaculture animals.

[0008] Xanthumol is a natural compound derived from the medicinal plant licorice. It has no toxic side effects on animals and humans and can be widely used in the preparation of drugs and feed additives for preventing Aeromonas hydrophila infection in aquatic animals.

[0009] Compared with the prior art, the present invention has the following advantages and beneficial effects: the anti-infective effect of xanthohumol is not to directly inhibit the growth of pathogens, so it will not cause selective pressure on pathogens and is not easy to produce drug resistance. In addition, since the drug does not inhibit bacteria, it still has a good therapeutic effect on drug-resistant bacteria. Xanthohumol is a natural compound that has no toxic effects on animals, humans and the ecological environment, and has a low risk of drug residue. Attached Figure Description

[0010] Figure 1 Growth curve of xanthohumol co-cultured with Aeromonas hydrophila strain XS-91-4-1

[0011] Figure 2 Effect of xanthohumol on the hemolytic activity of Aeromonas hydrophila culture supernatant

[0012] Figure 3 Effect of xanthohumol on the hemolytic activity of purified aerosols

[0013] Figure 4 Effects of xanthohumol on aerosol-induced A549 cell damage

[0014] Figure 5 The therapeutic effect of xanthohumol on Aeromonas hydrophila infection model Detailed Implementation

[0015] Example 1:

[0016] Determination of the minimum inhibitory concentration of xanthohumol against Aeromonas hydrophila

[0017] The Aeromonas hydrophila strain XS-91-4-1 involved in this invention was preserved by the Laboratory of Aquatic Animal Pharmacology and Drug Residue Control Technology, Yangtze River Fisheries Research Institute. The minimum inhibitory concentration (MIC) of xanthohumol against XS-91-4-1 was determined using the broth microdilution method recommended by CLSI. The steps are as follows:

[0018] (1) The prepared xanthohumol (Sichuan Weikeqi Biotechnology Co., Ltd., purity >98%, CAS No. 6754-58-1) was serially diluted in a 96-well cell culture plate to make the concentrations in wells 1 to 10 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL, respectively; the drug volume in each well was 100 μL.

[0019] (2) Aseptically inoculate single colonies of strain XS-91-4-1 into MH medium and incubate overnight at 30°C. Centrifuge the bacterial cells and adjust the concentration to 0.5 McFarland turbidity with sterile physiological saline, then dilute with medium to 1×10⁻⁶. 6 CFU / mL was added to a 96-well cell culture plate to achieve a final concentration of 5 × 10⁻⁶ CFU / mL. 5 CFU / mL was used to set up negative and positive control groups (negative control group received no drug or bacterial culture, positive control group received bacterial culture but no drug). Each drug was tested three times. After incubation in a 30℃ biochemical incubator for 18-24 hours, the results were observed. The lowest drug concentration at which no bacterial growth was observed was determined as the minimum inhibitory concentration (MIC) of the drug. The results showed that the MIC of xanthumol against Aeromonas hydrophila strain XS-91-4-1 was 128 μg / mL.

[0020] Example 2:

[0021] Effects of xanthohumol on the growth of Aeromonas hydrophila

[0022] The Aeromonas hydrophila strain XS-91-4-1 involved in this invention was preserved by the Laboratory of Aquatic Animal Pharmacology and Drug Residue Control Technology, Yangtze River Fisheries Research Institute. The XS-91-4-1 strain was cultured in BHI liquid medium until the early logarithmic growth phase (OD2). 600nm=0.3, 28℃), the cultured bacteria were placed in six 50mL Erlenmeyer flasks, 20mL in each flask, and different concentrations (0, 2, 4, 8, 16μg / mL) of xanthohumol were added to each flask. The flasks were incubated at 28℃ for 5 hours, and the OD was measured every 30 minutes. 600nm Absorption value. (Attached) Figure 1 The figure shows the growth curves of co-culture of different concentrations of xanthohumol with Aeromonas hydrophila strain XS-91-4-1. It can be seen from the figure that xanthohumol has no effect on the growth of Aeromonas hydrophila at a concentration of 16 μg / mL and below.

[0023] Example 3:

[0024] Effect of xanthohumol on hemolytic activity of Aeromonas hydrophila culture supernatant

[0025] The Aeromonas hydrophila strain XS-91-4-1 involved in this invention was preserved by the Laboratory of Aquatic Animal Pharmacology and Drug Residue Control Technology, Yangtze River Fisheries Research Institute. Aeromonas hydrophila XS-91-4-1 was cultured in BHI liquid medium to the early logarithmic growth phase. The bacterial suspension was then aliquoted into five 50mL Erlenmeyer flasks, 10mL in each flask. Xanthohumol was then added to each flask to achieve drug concentrations of 16, 8, 4, 2, and 0 μg / mL, respectively. The flasks were then cultured at 28℃ until the OD (Occurrence Limit) was reached. 600nm Centrifuge at 12000g for 1 min to collect the supernatant. Add trypsin to the supernatant and react at room temperature for 10 min to activate the aerolysins in the supernatant. Add 100 μL of activated supernatant, 875 μL of hemolysis buffer, and 25 μL of defibrinated sheep red blood cells to a 1.5 mL centrifuge tube, mix thoroughly, incubate at 37°C for 20 min, centrifuge at 10000g for 1 min, and measure the absorbance at OD543 nm of the supernatant. Deionized water was used as a positive control (100% hemolysis group), and hemolysis buffer was used as a negative control (0% hemolysis group). Results are attached. Figure 2 As shown, xanthohumol at concentrations of 4 μg / mL and above significantly reduced the hemolytic activity of Aeromonas hydrophila co-culture supernatant, and this inhibitory effect was dose-dependent. These results suggest that xanthohumol can reduce the hemolytic activity of culture supernatant by inhibiting aerolysin expression or activity.

[0026] Example 4:

[0027] Effect of xanthohumol on the hemolytic activity of Aeromonas hydrophila aerolysin

[0028] The aerosol involved in this invention was prepared and preserved by the Laboratory of Aquatic Animal Pharmacology and Drug Residue Control Technology, Yangtze River Fisheries Research Institute. The specific steps are as follows: 975 μL of PBS solution was added to five 1.5 mL centrifuge tubes, and 2 μL of purified aerosol at a concentration of 100 μg / mL was added to each tube. Then, xanthohumol was added to achieve concentrations of 0, 2, 4, 8, and 16 μg / mL in the different centrifuge tubes, respectively. After thorough mixing, the tubes were incubated at 37°C. After incubation for 15 min, 25 μL of defibrinated sheep erythrocytes (Shanghai Yuanye Biotechnology Co., Ltd.) were added, and incubation continued at 37°C for another 15 min. After high-speed centrifugation, the OD was measured. 543nm Absorbance was measured, with deionized water used as a positive control. (See attached image.) Figure 3 As shown, the hemolytic activity of aerosols decreases in a dose-dependent manner with increasing xanthohumol concentration. When the xanthohumol concentration reaches 2 μg / mL or higher, it can significantly inhibit aerosol activity. Combined with Examples 3 and 4, it can be seen that xanthohumol can reduce the hemolytic activity of Aeromonas hydrophila culture supernatant by directly inhibiting aerosol activity.

[0029] Example 5:

[0030] Protective effect of xanthohumol against A549 cell damage induced by Aeromonas hydrophila aerolysin.

[0031] The aerosol and A549 cells involved in this invention were preserved by the Yangtze River Fisheries Research Institute. A549 cells were cultured in an incubator containing 5% carbon dioxide in DMEM medium supplemented with 10% fetal bovine serum and penicillin-streptomycin antibiotics. During the experiment, the cells were cultured at a rate of 1.5 × 10⁶ cells / year. 5 Cells were seeded at a concentration of [number] cells / well in a 96-well cell culture plate and incubated overnight at 37°C in a CO2 incubator. The next day, 2 μL of purified aerosol at a concentration of 100 μg / mL was added to the culture plate, followed by the addition of xanthohumol to achieve final concentrations of 0, 2, 4, 8, and 16 μg / mL, respectively. After culturing the cells in a CO2 incubator for another 2 hours, the supernatant was collected, and the LDH release level in different treatment groups was measured using a lactate dehydrogenase (LDH) release kit. The results are attached. Figure 4 As shown, xanthohumol can dose-dependently reduce aerosol-induced LDH release from A549 cells, and significantly inhibit LDH release from A549 cells at concentrations of 2 μg / mL and above. These results indicate that xanthohumol can inhibit aerosol-induced A549 cell damage.

[0032] Example 6:

[0033] The therapeutic effect of xanthohumol on a crucian carp infection model of Aeromonas hydrophila.

[0034] The Aeromonas hydrophila strain XS-91-4-1 involved in this invention was preserved by the Laboratory of Aquatic Animal Pharmacology and Drug Residue Control Technology, Yangtze River Fisheries Research Institute. The experiment was conducted in a 100cm×50cm×60cm glass aquarium, with daily water changes and the water temperature maintained at approximately 28±2℃. Healthy crucian carp weighing approximately 100g were anesthetized and intraperitoneally injected with Aeromonas hydrophila strain XS-91-4-1 to establish an artificial infection model. Two hours post-infection, infected crucian carp were orally administered 20mg / kg of xanthohumol every 12 hours for 3 consecutive days. A positive control group was administered PBS after infection as a control. Twenty crucian carp were used in each experimental group. Mortality was recorded immediately after infection. Results are shown in the appendix. Figure 5 The results showed that the mortality rate of crucian carp artificially infected with Aeromonas hydrophila reached 80% within 8 days; 35% of crucian carp treated with 20 mg / kg xanthohumol died; analysis revealed that the survival rate of the xanthohumol treatment group was significantly higher than that of the positive control group. These studies indicate that crucian carp infected with Aeromonas hydrophila can be treated with 20 mg / kg xanthohumol, and this drug can achieve good therapeutic effects.

[0035] summary:

[0036] Aeromonas hydrophila is the most common pathogen in freshwater aquaculture, causing morbidity and mortality in various farmed aquatic animals and is a highly virulent disease in pond farming. Recent studies have found that the pathogenicity of Aeromonas hydrophila is closely related to its aerolysins, thus aerolysins have become a new target for research on drugs against Aeromonas hydrophila infection. Examples 1 and 2 of this invention showed that xanthohumol had almost no inhibitory effect on the growth of Aeromonas hydrophila strain XS-91-4-1; however, Examples 3 and 4 showed that xanthohumol could dose-dependently inhibit the hemolytic activity of Aeromonas hydrophila aerolysins. These examples indicate that xanthohumol can inhibit aerolysin activity at concentrations that do not inhibit the growth of Aeromonas hydrophila. Examples 5 and 6 investigated the inhibitory effect of xanthohumol on the in vitro and in vivo pathogenicity of Aeromonas hydrophila, finding that xanthohumol could reduce aerolysin-mediated A549 cell damage and improve the survival rate of a crucian carp infected with Aeromonas hydrophila. The above examples demonstrate that xanthumol can reduce the pathogenicity of Aeromonas hydrophila by inhibiting aerolysin activity, making it a potential natural compound for combating Aeromonas hydrophila infections. In conjunction with the above examples, the concentration at which xanthumol reduces aerolysin activity is far below its minimum inhibitory concentration (MIC), thus it is unlikely to induce drug resistance in Aeromonas hydrophila.

[0037] Aeromonas hydrophila has a wide host range, infecting various freshwater aquaculture organisms such as fish, frogs, and shrimp. Aerolysins are its hallmark virulence factors, widely present in pathogenic Aeromonas hydrophila. Therefore, it can be inferred that xanthumol has a therapeutic effect on infections caused by Aeromonas hydrophila.

Claims

1. Application of xanthohumol in the preparation of drugs for the prevention or treatment of Aeromonas hydrophila infection in aquatic animals.

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