Use of harman alkaloids for the preparation of pseudomonas plecoglossicida inhibitors

Abstract

The application discloses application of harmine in preparation of a pseudomonas plecoglossicida inhibitor, and is characterized by application of harmine in preparation of a medicine for preventing and / or treating infectious diseases of aquatic animals caused by the pseudomonas plecoglossicida, and further provides application of harmine or a pharmaceutically acceptable salt thereof in preparation of a medicine for preventing and treating pseudomonas plecoglossicida infection of aquatic animals, a medicine for preventing and treating liver injury of the pseudomonas plecoglossicida, an immune-regulating medicine for the pseudomonas plecoglossicida, and a feed additive for aquaculture; and has the advantages that the survival rate of aquatic animals can be significantly improved in a pseudomonas plecoglossicida infection model, bacterial load in blood and immune tissues such as liver, spleen and head kidney is reduced, and liver and spleen tissue pathological injury and cell apoptosis caused by infection are reduced; meanwhile, harmine can improve infection-related serum biochemical and antioxidant indexes, and has no obvious toxicity to fish cells within a certain concentration range.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to the application of halmanine in the preparation of an inhibitor for Pseudomonas aeruginosa. Background Technology

[0002] With the increasing scale and intensification of aquaculture, the frequency of bacterial diseases in aquatic animals has risen significantly, becoming a major factor affecting survival rates and economic benefits. Among these, diseases caused by *Pseudomonas* spp. (…) Pseudomonas Infectious diseases caused by bacteria are common in many farmed fish species and are difficult to control.

[0003] Ayubiosis ( Pseudomonas plecoglossicida (This is a common and important pathogen in aquaculture, which can infect economically important fish such as sweetfish, large yellow croaker, and grouper, causing visceral damage, increased inflammatory response, and high mortality, seriously affecting aquaculture yield and economic benefits. Due to the dense farming environment and rapid pathogen transmission, outbreaks often result in mass losses, posing a significant risk to the industry's safety.)

[0004] Currently, the prevention and control of Pseudomonas infections in aquatic animals mainly relies on antibiotics. However, the long-term or inappropriate use of antibiotics in aquaculture can easily lead to increased drug resistance in pathogens, reducing the effectiveness of prevention and control. At the same time, antibiotic residues also pose a potential risk to the quality and safety of aquatic products and the aquatic ecological environment. With the increasing demands for green aquaculture and food safety, the application of antibiotics in aquaculture is facing increasingly stringent restrictions. Therefore, developing safe, effective, low-residue, and non-resistant novel anti-infective drugs for fisheries has become one of the important research directions in the field of aquatic animal disease prevention and control.

[0005] Harmine is a β-carboline-based natural active small molecule with wide availability and certain biological activity. However, there is a lack of publicly available research reports and applications of harmine in the prevention and control of Pseudomonas aeruginosa infection in aquatic animals, and there is also a lack of technical solutions that can prove its in vivo preventive and therapeutic effects against this pathogen. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide an application of halmanine in the preparation of an inhibitor of *Pseudomonas aeruginosa*; the other is to provide an application of halmanine in the preparation of a drug for the prevention and / or treatment of infectious diseases in aquatic animals caused by *Pseudomonas aeruginosa*, thereby reducing mortality caused by infection and alleviating tissue damage.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: This invention provides an application of halmanine in the preparation of an inhibitor of Pseudomonas aeruginosa.

[0008] The present invention also provides the use of halmanine in the preparation of drugs for the prevention and / or treatment of infectious diseases in aquatic animals caused by Pseudomonas aeruginosa.

[0009] Preferably, the dosage of halmanine in the application is 10–200 μg / g body weight; more preferably, it is 50 μg / g or 100 μg / g body weight.

[0010] The present invention also provides the use of halmanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of Pseudomonas aeruginosa infection in aquatic animals.

[0011] The present invention also provides the use of halmanine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of Pseudomonas aeruginosa infection in aquatic animals.

[0012] Preferably, the aquatic animal is a fish; more preferably, the fish is ayu (Siniperca chuatsi). Plecoglossus altivelis ).

[0013] The present invention also provides the use of halmanine or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing liver injury in sweetfish.

[0014] The present invention also provides the use of halmanine or a pharmaceutically acceptable salt thereof in the preparation of an immunomodulatory drug for sweetfish.

[0015] The present invention also provides the use of halmanine or a pharmaceutically acceptable salt thereof in the preparation of aquaculture feed additives.

[0016] Compared with the prior art, the advantages of this invention are as follows: This invention discloses for the first time the application of harmine in the preparation of Pseudomonas aeruginosa inhibitors, which can significantly improve the survival rate of aquatic animals in a Pseudomonas aeruginosa infection models, reduce the bacterial load in blood and immune tissues such as liver, spleen, and head kidney, and alleviate the pathological damage and cell apoptosis of liver and spleen tissues caused by infection; at the same time, harmine can improve infection-related serum biochemical and antioxidant indicators, and has no obvious toxicity to fish cells within a certain concentration range, showing good application prospects. Attached Figure Description

[0017] Figure 1 The results of comparing the growth curves of 30 monomeric compounds derived from traditional Chinese medicine under 25 μM conditions in Specific Example 1 are as follows; Figure 2 Example 2 shows the effect of harmine on the in vitro antibacterial activity of Pseudomonas aeruginosa, where A represents the MIC-related inhibition results and B represents the effect of different concentrations of harmine on bacterial growth curves. Figure 3The following is a specific example of the inhibition results of harmine on biofilm formation of Pseudomonas aeruginosa in Example 3. A is a photograph of the biofilm amount determined by crystal violet staining, and B is a statistical graph of the biofilm inhibition rate. Figure 4 The following is a fluorescence staining result of the effect of harmine on the membrane permeability / membrane integrity of Pseudomonas aeruginosa in Specific Example 4. In this example, A is the fluorescence staining pattern of the bacterial membrane determined by the Hoechst / PI double staining method, and B is a statistical chart of the number of fluorescent staining bacteria in the bacterial membrane. Figure 5 This is a specific example of the effect of different doses of harmine treatment on the survival rate of sweetfish infected with Pseudomonas aeruginosa. Figure 6 The effect of different doses of harmine on the bacterial load in ayu after infection with Pseudomonas ayuensis is shown in Specific Example 6. In this example, A represents ayu blood, B represents liver, C represents spleen, and D represents head kidney tissue. Figure 7 The specific example seven illustrates the effects of different doses of harmine treatment on serum liver injury markers and antioxidant responses in ayu after infection with Pseudomonas ayuensis. AC represents the changes in the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), while DF represents the changes in the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content. Figure 8 For the specific example of the eighth embodiment, the effect of different doses of harmine treatment on the histopathological changes of sweetfish after infection with Pseudomonas aeruginosa was investigated. The samples were taken 12 h or 24 h after the last administration, where A was the liver and B was the spleen. Figure 9 For the specific example of the effect of different doses of harmine on the time-dependent and dose-dependent effects of apoptosis in ayu tissues after infection with Pseudomonas ayuensis, samples were taken 12 h or 24 h after the last administration. A is a TUNEL staining image of liver tissue and B is a TUNEL staining image of spleen tissue. Figure 10 In Specific Example 9, the mRNA expression levels of inflammation-related genes in the liver, spleen, and head kidney tissues of sweetfish were detected using the RT-qPCR method. Figure 11 For specific embodiments, the in vitro toxicity analysis of ten-halmanine on various normal cells is shown, where A is ayu macrophages (Ayu MO / Mф), B is L8824 cells, C is carp epithelioma cells (EPC), D is mouse mononuclear macrophage leukemia cells (Raw 264.7), E is mouse hepatocytes (AML12), and F is human embryonic kidney cells (HEK293T). Figure 12The effect of harmine on P. plecoglossicida-induced apoptosis in medaka Mo / Mф cells is shown in Figure A, where flow cytometry results of Mo / Mф cell apoptosis are presented, and figure B is a statistical graph of Mo / Mф cell apoptosis (n=3). In the above figure, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0019] Specific Implementation Example 1: Screening of Halmanine, a traditional Chinese medicine monomer with activity against Pseudomonas aeruginosa.

[0020] Killing the ayu pseudomonozoan ( P. plecoglossicida The frozen bacterial strain was revived and cultured overnight in TSB medium at 28 °C, and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 6 CFU / mL. Thirty monomeric compounds derived from traditional Chinese medicine with reported antibacterial activities were selected and prepared into working solutions with a final concentration of 25 μM. Each monomeric compound was added together with an equal volume of *Pseudomonas aeruginosa* bacterial suspension to a 96-well culture plate and incubated at 28 ℃. The optical density was measured every 2 h at 595 nm using a microplate reader to plot bacterial growth curves.

[0021] The results are as follows Figure 1 As shown, among the 30 monomers of traditional Chinese medicine tested, only halmanine showed a significant growth inhibitory effect on *Pseudomonas aeruginosa*, significantly prolonging its lag phase and significantly reducing its maximum growth density during the logarithmic growth phase; the other monomers had no significant effect on the growth of *Pseudomonas aeruginosa* under the same conditions. Therefore, halmanine was determined to be the core active ingredient with a significant inhibitory effect on *Pseudomonas aeruginosa*.

[0022] Specific Example 2: In vitro antibacterial effect of halmanine on *Pseudomonas ayuensis* and determination of minimum inhibitory concentration.

[0023] The bacterium *Pseudomonas aeruginosa* was revived and cultured overnight in TSB medium at 28°C, and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 8CFU / mL. 100 μL of bacterial culture was added to each well of a 96-well plate. Halamine dissolved in DMSO (final concentration <1%) was serially diluted 2-fold to achieve final concentrations of 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 μg / mL in the wells. A positive control group was prepared with 0.5 μg / mL ciprofloxacin (Cip.), and a blank control group was prepared with wells containing only the culture medium. Each concentration was tested in triplicate. After incubation at 28 ℃ and 150 rpm for 24 h, the optical density of each well was measured at 595 nm.

[0024] The results are as follows Figure 2 As shown in Figure A, halmanine exhibited a significant dose-dependent inhibitory effect on the growth of *Pseudomonas aeruginosa* within a concentration range of 20–200 μg / mL. With increasing halmanine concentration, bacterial survival gradually decreased. The half-maximal inhibitory concentration (MIC50) of halmanine against *Pseudomonas aeruginosa*, calculated by fitting a dose-response curve, was approximately 62.3 μg / mL. Further details are provided below. Figure 2 As shown in Figure B, at concentrations of halmanine of 20 μg / mL and 40 μg / mL, *Pseudomonas aeruginosa* still maintained significant growth, with its OD595 value showing no significant difference compared to the blank control group. However, when the halmanine concentration increased to 60 μg / mL and above, the growth curve of *Pseudomonas aeruginosa* became significantly flatter, and the OD595 value remained close to the baseline level, indicating that bacterial growth was significantly inhibited. Based on these results, it was determined that halmanine can significantly inhibit the growth rate of *Pseudomonas aeruginosa*, and that the maximum bacterial density reached during the logarithmic growth phase decreased in a dose-dependent manner with increasing drug concentration, indicating that halmanine can effectively inhibit the in vitro proliferation of this strain.

[0025] Specific Example 3: The inhibitory effect of halmanine on biofilm formation of Pseudomonas aeruginosa.

[0026] The bacterium *Pseudomonas aeruginosa* was revived and cultured overnight in TSB medium at 28°C, and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 6 CFU / mL, 100 μL was added to a 96-well plate. Halman alkali working solution was added to each well to a final concentration of 50 and 100 μg / mL, respectively; the control group only contained bacterial culture. The plates were incubated at 28 ℃ for 24 h. The supernatant was discarded, the plates were washed with PBS and air-dried, stained with 0.4% crystal violet for 20 min, washed again and air-dried, and the dye was dissolved in 33% glacial acetic acid. The absorbance was measured at 570 nm.

[0027] like Figure 3 A and Figure 3As shown in Figure B, in the control group, *Pseudomonas aeruginosa* formed a dense, continuous purple biofilm at the bottom of the culture wells. After the addition of halmanine, biofilm formation was significantly inhibited, and the degree of inhibition increased with increasing halmanine concentration. At 50 μg / mL, the biofilm thickness was significantly reduced; at 100 μg / mL, the biofilm structure showed significant fragmentation; and at 100 μg / mL, the biofilm almost completely disappeared, and the OD570 value decreased significantly. Based on these results, it is determined that halmanine can significantly inhibit biofilm formation of *Pseudomonas aeruginosa* in a dose-dependent manner, thereby disrupting its quorum defense structure and enhancing its sensitivity to external bactericidal agents.

[0028] Specific Example 4: Effect of Halmanine on Cell Membrane Permeability of Pseudomonas aeruginosa.

[0029] The bacterium *Pseudomonas aeruginosa* was revived and cultured overnight at 28°C in TSB medium, and the bacterial concentration was adjusted to 1×10⁻⁶. 6 CFU / mL. The bacterial culture was aliquoted into 6-well plates, 2 mL per well. Halmon alkaloids were added to the experimental groups at final concentrations of 50 μg / mL and 100 μg / mL, respectively. Ciprofloxacin was added to the positive control group at 0.5 μg / mL, and an equal volume of PBS was added to the control group. The plates were incubated at 28°C for 3 h. After incubation, the supernatant was discarded, and the plates were washed with PBS. The bacteria were stained for viability and mortality using the Hoechst / PI double staining method, observed under a fluorescence microscope, and photographed. The results are shown below. Figure 4 As shown.

[0030] like Figure 4 As shown in Figures A and 4B, the control group bacteria mainly exhibited blue fluorescence, indicating an intact cell membrane structure. After treatment with 50 μg / mL halmanine, some bacteria showed red fluorescence, indicating increased cell membrane permeability. After treatment with 100 μg / mL halmanine, a large number of bacteria showed red fluorescence, while blue fluorescence significantly decreased, with a trend similar to that of the ciprofloxacin-positive control group. These results indicate that halmanine can significantly increase the cell membrane permeability of *Pseudomonas aeruginosa*, disrupting its cell membrane integrity and thus exerting a direct bactericidal effect.

[0031] Specific Example 5: The therapeutic effect of halmanine on sweetfish infected with Pseudomonas aeruginosa.

[0032] One hundred and seventy-five healthy sweetfish, each measuring 5–10 cm in length, were selected and temporarily housed for 14 days to acclimatize to the experimental environment. The fish were randomly divided into five groups of 25 each. Except for the control group, all other groups were inoculated intraperitoneally with *Pseudomonas aeruginosa* at a dose of 5000 CFU / g body weight. Four hours post-infection, the fish were administered medications. The experimental groups were administered halometabolin via gavage at doses of 50 μg / g and 100 μg / g body weight, respectively; the positive control group was administered ciprofloxacin (10 μg / g body weight) via gavage; and the infected control group was administered an equal volume of DMSO via gavage. The survival rate of the sweetfish was observed and recorded daily for 14 consecutive days. Kaplan-Meier survival curves combined with log-rank tests were used to analyze the survival status of the sweetfish.

[0033] The results are as follows Figure 5 As shown, all ayu fish in the infected control group died on day 8 post-infection, with a survival rate of 0% on day 14. In contrast, the ayu fish treated with 50 μg / g and 100 μg / g halmanine showed survival rates of approximately 32% and over 40% on day 14, respectively, significantly higher than the infected control group and significantly better than the ciprofloxacin control group. These results indicate that halmanine can significantly improve the survival rate of ayu fish infected with *Pseudomonas ayuraceae*, and its preferred in vivo therapeutic dose is 50–100 μg / g body weight, preferably 100 μg / g.

[0034] Specific Example 6: The effect of halmanine on bacterial load in infected sweetfish.

[0035] Based on Specific Example 5, on day 7 post-infection, six surviving sweetfish from each group were randomly selected for sampling to detect bacterial load in vivo. Blood, liver, spleen, and head kidney tissue were collected from the sweetfish, weighed under aseptic conditions, homogenized with sterile PBS, serially diluted, and spread on tryptophan-soybean agar (TSA) plates. The plates were incubated at 28 °C for 24-48 h, and colony-forming units (CFU) were counted. The results are expressed as Log10 CFU / g.

[0036] The results are as follows Figure 6 A, Figure 6 B Figure 6 C and Figure 6 As shown in Figure D, the bacterial load in the blood, liver, spleen, and head kidney tissues of the infected control group was high. The bacterial load in all tissues of the ayu treated with 50 μg / g halmanine was significantly lower than that in the infected control group; the bacterial load in all tissues of the ayu treated with 100 μg / g halmanine was further reduced, approaching the levels of the uninfected blank control group, and significantly lower than that of the ciprofloxacin control group. These results indicate that halmanine can effectively eliminate *Pseudomonas ayuensis* in vivo, significantly reducing the bacterial load in the major immune organs of ayu, and its elimination effect is superior to that of conventional antibiotics.

[0037] Specific Example 7: Effects of Halmanine on Serum Biochemical Indicators of Infected Sweetfish.

[0038] Following the grouping and treatment methods in Specific Example 5, peripheral blood was collected from each group of sweetfish on day 7 post-infection. Serum was separated by centrifugation at 3000 r / min for 10 min. The activities of glutathione peroxidase (GSH-Px), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content were detected using commercially available kits.

[0039] The results are as follows Figure 7 As shown in Figure AF, the levels of AST, ALT, and ALP in the infected control group of sweetfish were significantly elevated, while the activities of SOD and CAT were significantly decreased, and the content of MDA was significantly increased, indicating that bacterial infection caused significant liver damage and oxidative stress. After treatment with halmanine, all abnormal indicators improved significantly. The levels of AST, ALT, and ALP in the 50 μg / g halmanine group were significantly lower than those in the infected control group; in the 100 μg / g halmanine group, these indicators further decreased and returned to levels close to those of the uninfected control group. At the same time, the activities of SOD and CAT were significantly increased, and the content of MDA was significantly decreased, which was better than that in the ciprofloxacin control group. These results indicate that halmanine can not only clear pathogens in the body, but also significantly alleviate liver function damage and oxidative stress imbalance caused by bacterial infection, and has a significant tissue protective effect on infected sweetfish.

[0040] Specific Example 8: Effects of Halmanine on Tissue Pathological Damage and Cell Apoptosis in Infected Sweetfish.

[0041] Ayu fish were infected and treated with medication according to the method in Specific Example 5. At 12 h and 24 h after the last administration, liver and spleen tissues were randomly sampled from each group of ayu fish, immediately fixed in 10% neutral formalin solution for 24-48 h, routinely embedded in paraffin, and sectioned to a thickness of approximately 4 μm. Some sections were stained with hematoxylin and eosin (H&E) to observe histopathological changes, while adjacent sections were stained with TUNEL to detect apoptosis levels.

[0042] The results are as follows Figure 8 A and Figure 8As shown in Figure B, the liver and spleen tissues of the infected control group of sweetfish showed significant pathological damage, manifested as hepatocyte degeneration and necrosis, disordered tissue structure, and extensive inflammatory cell infiltration, with significant destruction of the spleen white pulp structure. After treatment with halometadine, the above-mentioned pathological damage was significantly reduced. The degree of tissue damage was significantly reduced in the 50 μg / g halometadine group; the liver and spleen tissue structures in the 100 μg / g halometadine group were basically restored to normal, similar to the uninfected control group, and significantly better than the ciprofloxacin control group. TUNEL staining results. Figure 9 A and Figure 9 B showed that a large number of cells in the liver and spleen tissues of the infected control group underwent apoptosis; while the number of apoptotic cells was significantly reduced after treatment with halmanine, and the decrease showed a dose-dependent trend. These results indicate that halmanine can significantly alleviate the histopathological damage to sweetfish tissues caused by *Pseudomonas aeruginosa* infection and effectively inhibit infection-induced apoptosis, thereby protecting the host tissue structure and function.

[0043] Specific Example 9: The regulatory effect of halmanine on the expression of inflammatory-related factor genes in infected sweetfish.

[0044] Infection and drug administration were performed according to Specific Example 5. Liver, spleen and head kidney tissues of sweetfish were collected from each group at 12 h, 24 h and 48 h after drug administration. The mRNA expression levels of inflammation-related genes TNF-α, IL-1β, IL-6, IL-10, PRDX6 and HMGB1 were detected by real-time quantitative PCR, with β-actin as an internal control.

[0045] The results are as follows Figure 10 As shown in the figure, the mRNA expression levels of TNF-α, IL-1β, IL-6, PRDX6, and HMGB1 in various tissues of the infected control group of sweetfish were significantly upregulated, while IL-10 expression was significantly downregulated, indicating that bacterial infection induced a strong inflammatory response. After treatment with halmanine, the expression of various pro-inflammatory factors decreased significantly, and IL-10 expression was significantly upregulated. The 100 μg / g halmanine group showed the most significant regulatory effect, reaching a peak at 24 h after administration, which was superior to the ciprofloxacin control group. These results indicate that halmanine can significantly inhibit infection-induced excessive inflammatory response and promote the expression of anti-inflammatory factors, demonstrating a clear immunomodulatory effect.

[0046] Specific Example 10: Evaluation of the in vitro toxicity of halmanine to various normal cells.

[0047] Ayu macrophages, grass carp caudal fin cells (L8824), carp embryonic epithelioma cells (EPC), mouse macrophages (RAW264.7), mouse hepatocytes (AML12), and human embryonic kidney cells (HEK293T) were selected as test cell lines. Cells were seeded in 96-well plates and cultured to the logarithmic growth phase. Different concentrations of halmanine (0, 6.25, 12.5, 25, 50, 100, and 200 μg / mL) were added, and after 72 h of incubation, cell viability was detected using the CCK-8 assay.

[0048] The results are as follows Figure 11 As shown in Figure AF, at concentrations of halmanine at 100 μg / mL and below, the cell viability was not significantly different from the untreated control group; even at a high concentration of 200 μg / mL, only a slight inhibitory effect was observed. These results indicate that halmanine has essentially no significant toxicity to host cells within its effective antibacterial concentration range and possesses good safety.

[0049] Specific Example 11: The inhibitory effect of halmanine on apoptosis of infected ayu macrophages.

[0050] Ayu macrophages were infected with Pseudomonas ayuensis (MOI=10) for 1 h, and then incubated for 12 h with halmanine at concentrations of 50 μg / mL and 100 μg / mL, respectively. The apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry.

[0051] The results are as follows Figure 12 As shown in A and 12B, macrophages in the infection control group exhibited significant apoptosis, with a significantly increased total apoptosis rate. Treatment with halmanine significantly reduced the apoptosis rate, with a reduction of approximately 40% in the 50 μg / mL group and approximately 60% in the 100 μg / mL group, in a dose-dependent manner. These results indicate that halmanine can significantly inhibit bacterial infection-induced macrophage apoptosis and maintain the survival and function of immune cells.

[0052] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. Application of halmanine in the preparation of drugs for inhibiting Pseudomonas aeruginosa.

2. The use of halmanine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of Pseudomonas ayu infections in aquatic animals.

3. The use of halmanine or its pharmaceutically acceptable salt in the preparation of aquaculture feed additives for the prevention and / or treatment of Pseudomonas ayu infection in aquatic animals.

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