Use of chebulinic acid or chebulic acid in the preparation of a drug against water-borne pathogenic bacteria

Abstract

The application discloses application of chebulic acid or chebulinic acid in preparation of a medicine against aquatic pathogenic bacteria. The active tracking method is used to separate, purify and identify the active components in the extract of Terminalia chebula against Vibrio pathogenic bacteria, and the active components are identified as chebulic acid and chebulinic acid. The chebulic acid and the chebulinic acid are simple to prepare, have small toxic and side effects, and have strong bacteriostatic effects, especially on Vibrio parahaemolyticus. The experimental results show that the 96h half lethal concentrations of the chebulic acid and the chebulinic acid on fish are 561.653mg / L and 525.011mg / L respectively, which are 22.47 and 21.00 times of the minimum bacteriostatic concentration 25mg / L of the chebulic acid and the chebulinic acid on Vibrio parahaemolyticus, and the chebulic acid and the chebulinic acid are safe to fish and have broad-spectrum bacteriostatic effects on Pseudomonas aeruginosa, Aeromonas veronii, Aeromonas hydrophila, Vibrio alginolyticus, Vibrio vulnificus and Vibrio parahaemolyticus, and have good application prospects in the field of new environment-friendly fish medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an application of chebulagic acid or chebulinic acid in preparation of a drug for resisting aquatic pathogenic bacteria. BACKGROUND

[0002] Aquatic pathogenic microorganisms are the main source of water product safety problems and diseases, which not only brings huge economic losses to global aquaculture, but also poses a threat to human health, especially some pathogenic Vibrio. In recent years, China's aquaculture industry has developed rapidly, and the fishery output has increased significantly, ranking among the world's top. However, with the continuous increase of aquaculture scale and intensification, diseases caused by aquatic pathogenic bacteria occur frequently, which not only affects the quality and safety of aquatic products, but also poses a challenge to the sustainable development of aquaculture industry.

[0003] At present, chemical drugs are mostly used to prevent and treat aquatic bacterial diseases, such as using antibiotic drugs. The large and long-term use of traditional antibiotics has produced a large number of drug-resistant strains, which not only increases the difficulty of preventing and treating bacterial diseases, but also poses a threat to food safety and public health. Chinese herbal medicine has become a potential substitute for antibiotics due to its green, safe, small toxic and side effects, and not easy to produce drug resistance. In addition, in order to reduce the use of antibiotics, the Ministry of Agriculture and Rural Affairs promotes the action of reducing the use of veterinary antibacterial drugs, encourages the use of veterinary Chinese medicine, microecological preparations and other non-residual green veterinary drugs to replace part of veterinary antibacterial drugs, in order to promote the green development of aquaculture and the safety of aquatic products.

[0004] Chebulagic acid and chebulinic acid are both derived from Terminalia chebula. The structure of the compound has been disclosed in the literature (Gunawan-Puteri, Maria et al. Novel alpha-glucosidase inhibitors from Macaranga tanarius leaves. Food Chemistry, 2010, 123(2): 384-389.), and the structural formula of chebulagic acid is as follows:

[0005]

[0006] The molecular formula is: C 41 H 30 O 27 , and the molecular weight is 954.66. It is a white amorphous powder, easily soluble in chloroform, methanol, acetone, ethanol and other organic solvents.

[0007] The structural formula of chebulinic acid is as follows:

[0008]

[0009] The molecular formula is: C 41 H32 O 27 , molecular weight is 956.68, white amorphous powder. Easily soluble in chloroform, methanol, acetone, ethanol and other organic solvents.

[0010] Chebulic acid, Terminalia chebula Retz. And its variety of Terminalia chebula Retz. The dry mature fruit of tomentella Kurt. Distributed in Tibet, Yunnan, Guangdong, Guangxi and other places in China. Chebulic acid has antioxidant, anti-inflammatory, antitumor, anti-pathogenic microorganism, hypoglycemic, anti-obesity, prevention of cardiovascular disease and other various pharmacological activities, and has great medicinal development value. There is no report that active ingredients of chebulic acid and chebulic acid have anti-aquatic pathogenic bacteria effect. SUMMARY

[0011] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a kind of application of chebulic acid or chebulic acid in the preparation of anti-aquatic pathogenic bacteria drug. Chebulic acid and chebulic acid in chebulic acid extract have small side effects and strong bacteriostatic effect, especially in inhibiting Vibrio parahaemolyticus.

[0012] The present application adopts active tracking method to separate, purify and identify the active ingredients of chebulic acid extract against pathogenic Vibrio, and identifies chebulic acid and chebulic acid. Further research shows that the active ingredients in chebulic acid can inhibit a variety of aquatic pathogenic bacteria.

[0013] The purpose of the present application is realized by the following technical scheme:

[0014] The application of chebulic acid or chebulic acid or its pharmaceutically acceptable salt in the preparation of anti-aquatic pathogenic bacteria drug or feed additive;

[0015] The application of chebulic acid or chebulic acid or its pharmaceutically acceptable salt in the preparation of anti-aquatic pathogenic bacteria infection drug or feed additive;

[0016] The aquatic pathogenic bacteria is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas veronii and Aeromonas hydrophila. Further, it is Vibrio parahaemolyticus.

[0017] The chebulic acid or chebulic acid can be prepared by various methods known in the art or modified methods from plants, synthesized or semi-synthesized, or directly purchased from market.

[0018] Further, the chebulic acid or chebulic acid is obtained by extraction and separation from chebulic acid.

[0019] The drug or feed additive comprises a therapeutically effective amount of chebulic acid or chebulic acid or its pharmaceutically acceptable salt.

[0020] The MIC value of chebulic acid or chebulinic acid to Vibrio parahaemolyticus is 25 mg / L, and the safe concentration of chebulic acid to fish is in the range of 25 mg / L to 143 mg / L, and the safe concentration of chebulinic acid to fish is in the range of 25 mg / L to 169 mg / L.

[0021] Further, the safe concentration of chebulic acid is 143.21 mg / L, and the safe concentration of chebulinic acid is 169.27 mg / L.

[0022] The medicine is prepared into a medicinal liquid or a powder.

[0023] The medicine contains one or more pharmaceutically acceptable carriers or excipients.

[0024] The chebulic acid and chebulinic acid of the present application can inhibit Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas veronii and Aeromonas hydrophila. Therefore, the antibacterial activity of chebulic acid and chebulinic acid has a good application prospect in the field of new environmentally friendly fishery drugs.

[0025] The present application has the following advantages and effects relative to the prior art:

[0026] (1) The present application finds a new, efficient and non-polluting drug resource for resisting aquatic pathogenic bacteria aiming at the problem of pathogenic bacteria of Vibrio in current aquaculture.

[0027] (2) The present application first uses chebulic acid and chebulinic acid to prepare a drug for resisting aquatic pathogenic bacteria, especially for inhibiting Vibrio parahaemolyticus.

[0028] (3) The raw material of the method of the present application is abundant, chebulic myrobalan is a traditional Chinese medicine in China and is easy to obtain, the chebulic myrobalan extract is obtained by extracting chebulic myrobalan with anhydrous ethanol as a solvent, and it is determined that the effective components of chebulic myrobalan for inhibiting aquatic pathogenic bacteria are chebulic acid and chebulinic acid; and chebulic acid and chebulinic acid are simple to prepare and have a wide application prospect.

[0029] (4) The MIC value of chebulic acid and chebulinic acid of the present application to Vibrio parahaemolyticus is 25 mg / L, the 96h semi-lethal concentration to fish is 561.653 mg / L and 525.011 mg / L respectively, which is 22.47 and 21.00 times of the minimum inhibitory concentration 25 mg / L of Vibrio parahaemolyticus, is safe to fish, and has a broad-spectrum antibacterial effect, and can inhibit Pseudomonas aeruginosa, Aeromonas veronii, Aeromonas hydrophila, Vibrio alginolyticus, Vibrio vulnificus and Vibrio parahaemolyticus. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the C-NMR spectrum of chebulic acid. 13 C-NMR spectrum.

[0031] Figure 2 is the Terminalia acid 1 H-NMR spectrum.

[0032] Figure 3 is the Terminalia acid 13 C-NMR spectrum.

[0033] Figure 4 is the Terminalia acid 1 H-NMR spectrum.

[0034] Figure 5 is the Terminalia acid HPLC.

[0035] Figure 6 is the Terminalia acid HPLC.

[0036] Figure 7 is the Terminalia acid antibacterial experiment on Vibrio parahaemolyticus (mg / L).

[0037] Figure 8 is the OD value of Vibrio parahaemolyticus treated by Terminalia acid at different concentrations; Note: compared with before culture, *p<0.05, **p<0.01.

[0038] Figure 9 is the Terminalia acid antibacterial experiment on Vibrio parahaemolyticus (mg / L).

[0039] Figure 10 is the OD value of Vibrio parahaemolyticus treated by Terminalia acid at different concentrations; Note: compared with before culture, *p<0.05, **p<0.01.

[0040] Figure 11 is the growth curve of Vibrio parahaemolyticus treated by Terminalia acid.

[0041] Figure 12 is the growth curve of Vibrio parahaemolyticus treated by Terminalia acid.

[0042] Figure 13 is the AKP activity of Vibrio parahaemolyticus treated by Terminalia acid; Note: compared with CK, *p<0.05, **p<0.01.

[0043] Figure 14 is the AKP activity of Vibrio parahaemolyticus treated by Terminalia acid; Note: compared with CK, *p<0.05, **p<0.01.

[0044] Figure 15 is the morphological change of Vibrio parahaemolyticus under the action of different concentrations of Terminalia acid; among them, a: CK; b: 1 / 2MIC; c: 1MIC. DETAILED DESCRIPTION

[0045] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0046] As a preferred solution, the chebulagic acid or chebulinic acid has an activity against the aquatic pathogenic bacteria Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas veronii and Aeromonas hydrophila.

[0047] As a preferred solution, the chebulagic acid or chebulinic acid is safe for fish in the effective bacteriostatic concentration range of 25-143 mg / L, and has an effect against the infection of aquatic pathogenic bacteria.

[0048] As a preferred solution, the chebulagic acid or chebulinic acid can be prepared by various methods known in the art or modified methods, or can be obtained by extraction, synthesis or semi-synthesis from plants, or can be directly purchased from the market.

[0049] As a preferred solution, the chebulagic acid or chebulinic acid is obtained by extraction and separation from the fruits of Terminalia chebula.

[0050] The specific preparation method is as follows:

[0051] Dry 2 kg of Terminalia chebula fruits, and extract 2 times with 20 L of 90% ethanol by reflux extraction, each time for 2 hours. Combine the extract, and concentrate to obtain a total extract of 440 g. After the total extract is suspended with water (m:v = 1 g:1 mL), sequentially extract with petroleum ether and ethyl acetate to obtain a petroleum ether part of 110 g and an ethyl acetate part of 280 g. After an antibacterial experiment, it is determined that the ethyl acetate part has an activity. The ethyl acetate part is subjected to HP20 macroporous resin column chromatography, and gradient elution is performed with 10%, 20%, 30%, 40%, 50%, 70%, 90% and 100% ethanol as the elution system, and each level of eluent is 12 L. According to the antibacterial activity of each part of the macroporous resin column chromatography, the 20% ethanol eluate is subjected to preparative high-performance liquid chromatography, gradient elution is performed with methanol:water (15%-60%) as the mobile phase for 90 min, the flow rate is 5 ml / min, and the eluent is 2 L, to obtain active compound 1, which is identified as chebulagic acid ( 13 The C-NMR spectrum is shown in Figure 1 , 1 The H-NMR spectrum is shown in Figure 2 ). The 30% ethanol eluate is subjected to preparative high-performance liquid chromatography, gradient elution is performed with methanol:water (20%-50%) as the mobile phase for 90 min, the flow rate is 5 ml / min, and the eluent is 2 L, to obtain active compound 2, which is identified as chebulinic acid ( 13 The C-NMR spectrum is shown in Figure 3 , 1 The H-NMR spectrum is shown in Figure 4 ).

[0052] The prepared chebulic acid and chebulinic acid can be detected by the following method:

[0053] Determination of purity of chebulic acid and chebulinic acid by HPLC

[0054] 1 mg of chebulic acid and chebulinic acid was precisely weighed and placed in a 1 mL sample bottle, 1 mL of methanol was added, and ultrasonic oscillation was used for dissolution. The sample purity was determined by normalization method. The liquid chromatograph was Agilent 1100 series; Welch Ultimate XB-C18, 4 mm x 250 mm, 5 μm was used as the chromatographic column; the mobile phase A was 0.1% phosphoric acid solution in water; the mobile phase B was analytical grade methanol; gradient elution (0-30 min, 15%→60% B) was used; the volume flow rate was 1 mL / min; the column temperature was 30°C; the sample injection amount was 10 μL; and the detection wavelength was 270 nm.

[0055] The high performance liquid chromatogram of chebulic acid is shown in Figure 5 The results show that there is only one single peak without other impurity peaks, i.e., the purity of chebulic acid is relatively high, and the impurities are very few. The purity of chebulic acid reaches more than 98%.

[0056] The high performance liquid chromatogram of chebulinic acid is shown in Figure 6 The results show that there is only one single peak without other impurity peaks, i.e., the purity of chebulinic acid is relatively high, and the impurities are very few. The purity of chebulinic acid reaches more than 98%.

[0057] Example 1: Determination of MIC value of chebulinic acid and chebulic acid on Vibrio parahaemolyticus

[0058] (1) Preparation of chebulinic acid and chebulic acid liquid medicine: 10 mg of chebulinic acid and chebulic acid was weighed on a ten-thousandth balance, respectively, and dissolved in 1 mL of ultrapure water containing 0.5% DMSO to obtain a 10000 mg / L primary stock solution. 0.1 mL of the primary stock solution and 0.9 mL of ultrapure water were taken to obtain 1 mL of a 1000 mg / L secondary stock solution. Then, using a two-fold gradient dilution method, 1 mL of 500, 250 mg / L drug solution was prepared, and using a ten-fold gradient dilution method, 1 mL of 100, 50, 25 mg / L drug solution was prepared. At this time, the DMSO content of the 1000 mg / L drug solution was 0.05%, and the DMSO content of the 0 mg / L (control group) drug solution was also prepared to be 0.05%.

[0059] (2) Bacterial solution preparation: 100 μL of Vibrio parahaemolyticus strain in glycerol tube stored in -80 °C refrigerator was inoculated into 10 mL of LB liquid medium, and cultured at 28 °C with 200 rpm shaking for 12 h overnight activation to make the strain in late exponential growth phase. The absorbance value of the bacterial solution at 600 nm was measured, and the concentration of each bacterial solution was adjusted to 1 x 107~ 1 x 10 8 CFU / mL.

[0060] (3) In a sterile environment, 100 μL of diluted bacterial solution and 100 μL of prepared drug solution were added to a 96-well plate to make the final concentration 500, 250, 125, 50, 25, 12.5 mg / L, respectively. Three parallel groups were set for each experiment. The plate was incubated at 28 °C for 12 h. The bacterial solution without adding chebulagic acid or chebulinic acid was used as negative control (CK), and the bacterial solution with added antibiotic (ampicillin, final concentration 2500 mg / L) was used as positive control (A+). The absorbance value at 600 nm was measured, and the MIC was the minimum concentration of chebulagic acid and chebulinic acid when the bacterial cells in the plate were not visible to the naked eye.

[0061] The results are shown as follows: From Figure 7 and Figure 8 It can be seen that the MIC value of Vibrio parahaemolyticus treated with chebulagic acid alone for 12 h was 25 mg / L, which showed that chebulagic acid had an inhibitory effect on the growth of Vibrio parahaemolyticus. Figure 9 and Figure 10 It can be seen that the MIC value of Vibrio parahaemolyticus treated with chebulagic acid alone for 12 h was 25 mg / L, which showed that chebulagic acid had an inhibitory effect on the growth of Vibrio parahaemolyticus.

[0062] Example 2: Effect of chebulagic acid and chebulinic acid on the growth curve of Vibrio parahaemolyticus

[0063] (1) Preparation of chebulagic acid and chebulinic acid drug solution: According to the method of Example 1, 30 mL of drug solution was prepared at concentrations of 25, 50, 100 and 0 (control) mg / L.

[0064] (2) Bacterial solution preparation: According to the method of Example 1, 80 mL of Vibrio parahaemolyticus bacterial solution was prepared at concentrations of 1 x 107~ 1 x 10 8 CFU / mL.

[0065] (3) In a sterile environment, 10 mL of diluted bacterial solution and 10 mL of prepared drug solution were added to the test tube respectively, so that the final concentration was 12.5 (1 / 2 MIC), 25 (1 MIC), 50 (2 MIC) mg / L respectively, and three parallel groups were set for each experiment. Incubate at 28°C, 200 rpm for 24 h, take sample every 2 h, use bacterial solution without chebulagic acid or chebulinic acid as negative control, measure the absorbance value at 600 nm, and draw the growth curve.

[0066] The results are as follows: Figure 11 and Figure 12 It can be seen that with the increase of the concentration of chebulagic acid and chebulinic acid, the growth of Vibrio parahaemolyticus gradually weakens. In the 1 / 2 MIC chebulagic acid or chebulinic acid treatment group, the growth trend of Vibrio parahaemolyticus is obviously slowed down compared with the control group, and in the 1 MIC and 2 MIC chebulagic acid or chebulinic acid treatment group, the growth curve is flat and no obvious bacterial growth occurs.

[0067] Example 3: Antibacterial effect of chebulagic acid and chebulinic acid on other pathogenic bacteria

[0068] (1) Preparation of chebulagic acid and chebulinic acid solution: According to the method of Example 1, 1 mL of 5000, 2500 and 1250 mg / L drug solution was prepared.

[0069] (2) Preparation of bacterial solution: According to the method of Example 1, 1 mL of bacterial solution with a concentration of 1×107-1×10 8 CFU / mL of Pseudomonas aeruginosa, Aeromonas veronii, Aeromonas hydrophila, Vibrio alginolyticus, Vibrio vulnificus or Vibrio parahaemolyticus was prepared.

[0070] (3) The inhibitory effect of chebulagic acid and chebulinic acid on pathogenic bacteria was determined by agar diffusion method. In a sterile environment, 0.1 mL of bacterial solution was taken with a pipette and evenly coated on the solid culture medium. When there is no liquid flow, use a puncher (diameter 6 mm) to punch three evenly distributed holes on the solid culture medium. Two holes are added with 100 μL of drug solution, and the other hole is added with 100 μL of liquid culture medium (without drug) as control. Three parallel groups were set for each experiment. Then the culture dish was placed in a 28°C constant temperature incubator for 12 h, and the diameter of the inhibition zone (mm) was measured and recorded. The results are shown in Tables 1 and 2.

[0071] Table 1 Diameter of chebulagic acid inhibition zone on other pathogenic bacteria (mm)

[0072]

[0073] Table 2 Diameter of chebulinic acid inhibition zone on other pathogenic bacteria (mm)

[0074]

[0075] From the results of Table 1, Table 2, it can be seen that: it can be seen that, acacia acid or chebulic acid has inhibitory effect on pseudomonas aeruginosa, aeromonas veronii, aeromonas hydrophila, vibrio alginolyticus, vibrio vulnificus, vibrio parahaemolyticus.

[0076] Example 4: Effect of acacia acid and chebulic acid on the activity of alkaline phosphatase (AKP) of vibrio parahaemolyticus

[0077] (1) Preparation of acacia acid and chebulic acid liquid medicine: according to the method of example 1, 25, 50, 100 and 0 (control) mg / L of liquid medicine each 3mL.

[0078] (2) Preparation of bacterial solution: according to the method of example 1, prepare 50mL of vibrio parahaemolyticus solution with concentration of 1×107~1×10 8 CFU / mL.

[0079] (3) In sterile environment, add 1mL of diluted bacterial solution and 1mL of prepared drug solution in test tube respectively, so that the final concentration is 12.5 (1 / 2MIC), 25 (1MIC), 50 (2MIC) mg / L, and set three groups of parallel for each experiment. 28℃, 200rpm for 10h, with no acacia acid or chebulic acid added as negative control (CK). Take 1mL of bacterial solution, centrifuge at 12000rpm for 1 minute, take the supernatant, and use alkaline phosphatase (AKP) kit to determine the AKP content. Each sample is repeated three times.

[0080] The results are as follows: from Figure 13 and Figure 14 It can be seen that, after treatment of acacia acid or chebulic acid, the extracellular AKP activity of different concentrations of vibrio parahaemolyticus is extremely significantly increased compared with the control group (p<0.01), and it is enhanced with the increase of concentration, which shows that acacia acid or chebulic acid can destroy the cell wall integrity of vibrio parahaemolyticus.

[0081] Example 5: Effect of acacia acid on the morphology of vibrio parahaemolyticus

[0082] (1) Preparation of acacia acid liquid medicine: according to the method of example 1, prepare 12.5 (1 / 2MIC), 25 (1MIC) mg / L and 0 (control) mg / L of liquid medicine each 1mL.

[0083] (2) Preparation of bacterial solution: according to the method of example 1, prepare 2mL of vibrio parahaemolyticus solution with concentration of 1×107~1×10 8 CFU / mL.

[0084] (3) Drug treatment: Place a layer of cellophane on the LB solid plate, spread 100 μL of bacterial solution with a sterile spreader, incubate in a 37°C incubator for 3 h, and then remove and set aside. Cut the cellophane into 1 × 1 cm pieces and soak them in solutions with 1 / 2 MIC, 1 MIC, and a control solution for 6 h. The cellophane sample without drug treatment was designated as the control (CK).

[0085] (4) Fixation: Fix in 2.5% (V / V) glutaraldehyde solution for 1.5 h.

[0086] (5) Dehydration treatment: The samples were placed in 30%, 50%, 70%, 90%, and 100% alcohol for dehydration, with each concentration treated for 10 minutes.

[0087] (6) Drying: Dry in a low-temperature freeze dryer for 1 hour.

[0088] (7) After drying, take out the cellophane sample and stick it on the copper table with double-sided tape. After spraying gold, observe and photograph it under a scanning electron microscope.

[0089] The results are as follows: Figure 15 As can be seen in a, the Vibrio parahaemolyticus cells not treated with cheleic acid showed a typical Vibrio structure with a complete, smooth, short rod-like appearance; Figure 15 As can be seen in b, when exposed to 1 / 2MIC cheleic acid, obvious depressions appeared on the cell surface and the morphology was abnormal; Figure 15 As can be seen from c in Figure 1, when the concentration is 1MIC, Vibrio parahaemolyticus cells show holes and lysis, with no complete cell structure and leakage of intracellular substances.

[0090] Example 6: Safety assessment of chebulic acid and chebulic acid on goby

[0091] Preliminary experiments determined that the drug concentrations of chebulic acid and chebulic acid that caused all deaths of gobies within 24 hours were both 1000 mg / L, and the concentrations that did not cause deaths within 96 hours were both 100 mg / L.

[0092] On the basis of the preliminary experiment, five concentration groups of 800, 500, 250, 100 and 0 (control) mg / L were set up, with three parallels for each concentration. 27 buckets (volume: 10L) were filled with 2L of aerated 25‰ seawater and 8 gobies, the temperature was controlled at 23±0.5℃, and oxygen was supplied by an oxygen supply pump. During the experiment, the poisoning of gobies in each group was observed every day. If gobies were found to have died of poisoning, they should be fished out in time to avoid damaging the water quality and affecting the experiment. After 96h, the experimental results were statistically analyzed and the half-lethal concentration (LC50) was calculated. 50) and 95% confidence interval (calculated using SPSS piece probit procedure), as shown in Table 3, Table 4. Safety concentration = 48h LC 50 x 0.3 / (24h LC 50 / 48h LC 50 ) 2 .

[0093] Table 3 Acute toxicity test results of chebulagic acid and chebulinic acid on Odontesthes bonariensis

[0094]

[0095] Table 4 Median lethal concentration (LC 50 ) of chebulagic acid and chebulinic acid on Odontesthes bonariensis

[0096]

[0097] The results are as follows: As can be seen from Table 3, the survival rate of Odontesthes bonariensis in the 100 and 0 mg / L treatment groups was 100%, and the mortality rate increased with increasing drug concentration, and in the 1000 mg / L treatment group, all Odontesthes bonariensis died within 24h. As can be seen from Table 4, the 96h-LC 50 of chebulagic acid on Odontesthes bonariensis was 561.653 mg / L, and the safety concentration was 143.21 mg / L, and the 96h-LC 50 of chebulinic acid on Odontesthes bonariensis was 525.011 mg / L, and the safety concentration was 169.27 mg / L. This shows that the two active compounds are safe for fish in the effective antibacterial concentration range of 25-143 mg / L, and can be applied to farmed fish.

[0098] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Use of chebulic acid or chebulic acid as the sole active ingredient in the preparation of a drug for resisting aquatic pathogens, characterized in that: The aquatic pathogenic bacteria is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas versii and Aeromonas hydrophila.

2. Use of chebulic acid or chebulic acid as the sole active ingredient in the preparation of a medicament for resisting aquatic pathogen infection, characterized in that: The aquatic pathogenic bacteria is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas versii and Aeromonas hydrophila.

3. Use of chebulic acid or chebulic acid as the sole active ingredient in the preparation of a feed additive for resisting aquatic pathogens, characterized in that: The aquatic pathogen is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas veseri and Aeromonas hydrophila; the safe concentration of chebulic acid is 143.21 mg / L, and the safe concentration of chebulic acid is 169.27 mg / L.

4. Use of chebulic acid or chebulic acid as the sole active ingredient in the preparation of a feed additive for resisting aquatic pathogen infection, characterized in that: The aquatic pathogen is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Pseudomonas aeruginosa, Aeromonas veseri and Aeromonas hydrophila; the safe concentration of chebulic acid is 143.21 mg / L, and the safe concentration of chebulic acid is 169.27 mg / L.

5. The use according to any one of claims 1 to 4, characterized in that: The aquatic pathogen is Vibrio parahaemolyticus.

6. The use according to any one of claims 1 to 4, characterized in that: The chebulic acid or chebulic acid is prepared by separation, extraction, synthesis or semi-synthesis from plants.

7. The use according to claim 6, characterized in that: The chebulic acid or chebulic acid is obtained by extraction and separation from Terminalia chebula.

8. The use according to claim 1 or 2, characterized in that: The safe concentration of chebulic acid is 143.21 mg / L, and the safe concentration of chebulic acid is 169.27 mg / L.

9. The use according to claim 1 or 2, characterized in that: The medicine is prepared into liquid or powder.

10. The use according to claim 1 or 2, characterized in that: The medicine contains one or more pharmaceutically acceptable carriers or excipients.

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