Chinese herbal medicine sappan wood for treating tilapia bacterial meningitis, application and application method

Abstract

The invention discloses Chinese herbal medicine sappan wood for treating tilapia bacterial meningitis, application and an application method, the Chinese herbal medicine sappan wood effectively becomes brazilin, the brazilin is a homoisoflavone compound, and the molecular formula of the brazilin is C16H14O5. The application includes that the compound has an inhibiting effect on tilapia mossambica source streptococcus agalactiae and can penetrate through a tilapia mossambica blood brain barrier. The application method comprises the steps of soaking treatment, pouring treatment and mixing treatment. The hematoxylon is selected as the medicine for treating the nile tilapia bacterial meningitis for the first time and has the effects of lowering the expression level of inflammatory factors of the nile tilapia bacterial meningitis and improving the survival rate of the nile tilapia, and the Chinese herbal medicine hematoxylon used in the invention is simple in use mode and low in price and has very strong clinical transformation significance.

Description

Technical Field

[0001] The present invention relates to the technical field of fish meningitis treatment, and particularly to Chinese herbal medicines sappanwood for treating tilapia bacterial meningitis, their applications and application methods. Background Art

[0002] Streptococcus agalactiae can infect a variety of aquatic animals, among which tilapia shows extremely high susceptibility. Streptococcus agalactiae can break through the blood-brain barrier and invade the central nervous system, leading to tilapia bacterial meningitis and neurological dysfunction. The typical clinical features of diseased tilapia are blackening of the body surface, exophthalmos, corneal opacity, and neurological motor disorders. After dissection, relevant pathological changes such as inflammatory exudation and bacterial masses in the brain can be observed. The mortality rate of diseased tilapia is extremely high. Currently, the prevention and treatment mainly rely on antibiotics. However, the long-term abuse of antibiotics will not only lead to the production of drug resistance in pathogenic bacteria but also form drug residues in fish, threatening food safety. Therefore, it is crucial to find a drug that can effectively reduce the degree of tilapia bacterial meningitis and the mortality rate.

[0003] The first consideration in screening drugs for treating bacterial meningitis is whether the drug can penetrate the blood-brain barrier. The blood-brain barrier is a crucial dynamic separation interface between the blood and the brain tissue, strictly controlling the exchange of substances between the blood and the brain tissue. Under normal physiological conditions, only oxygen, carbon dioxide, and small-molecule lipophilic substances can pass through the blood-brain barrier by free diffusion. The blood-brain barrier also has an active efflux system that can selectively pump harmful substances and excess metabolites in the brain out of the brain and prevent exogenous harmful substances from entering the brain tissue to maintain the homeostasis of the brain tissue. The structural basis and functional core of the blood-brain barrier are the tight junctions formed at the apical ends of brain microvascular endothelial cells, which have high structural complexity and functional stability. Their main function is to maintain cell polarity and form a barrier to prevent ions and solutes from passing through the cell gaps, thereby maintaining the mechanical barrier and permeability between cells. The main components of tight junction proteins include transmembrane proteins, cytoplasmic attachment proteins, and cytoskeletal proteins. Under the category of transmembrane proteins, there are also occludin, claudin, and junctional adhesive molecule (JAM); ZO-1 (zonula occludens-1) is a cytoplasmic attachment protein. In the treatment of many brain-derived diseases, Chinese herbal medicines have shown good curative effects. Their treatment mechanisms may involve two aspects: one is that the active ingredients directly penetrate the blood-brain barrier and act on the central nervous system; the other is to exert curative effects by regulating the structure and function of the blood-brain barrier.

[0004] Studies have shown that if Chinese herbal medicines contain lipophilic small molecule substances, they are very likely to penetrate the blood-brain barrier and play a therapeutic role in the brain tissue; at the molecular level, the activity of some Chinese herbal medicines can play a therapeutic role by regulating the expression level of tight junction proteins to increase the permeability of the blood-brain barrier.

[0005] Therefore, it is of great significance to provide a Chinese herbal medicine sappanwood, its application and application method, which have the effect of down-regulating the expression level of inflammatory factors in tilapia bacterial meningitis and improving the survival rate of tilapia. Summary of the Invention

[0006] In view of this, the present invention provides a Chinese herbal medicine sappanwood, its application and application method for treating tilapia bacterial meningitis.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis, the active ingredient of the Chinese herbal medicine sappanwood is brazilein, and the brazilein is a high isoflavone compound with the molecular formula C 16 H 14 O5.

[0009] Preferably, the concentration ratio of the extract of the Chinese herbal medicine sappanwood to the concentrated Chinese herbal medicine sappanwood is 10:1.

[0010] Preferably, the LC / MS detection method of the active ingredient brazilein is as follows:

[0011] Electrospray ionization source ESI, detected in positive ion mode, and the scanning mode is selected as multiple reaction monitoring MRM; mobile phase A: acetonitrile - B: 0.2% formic acid, detection time: 15 min, gradient elution 0 - 3 min, A: 35 → 10, B: 65 → 90; 6 - 6.5 min, A: 10 → 35, B: 90 → 65, flow rate 200 μL / min, injection volume 5 μL.

[0012] The application of the Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis, the application includes: having an inhibitory effect on Streptococcus agalactiae from tilapia and being able to penetrate the blood-brain barrier of tilapia.

[0013] The application method of the Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis, the method includes: immersion treatment, gavage treatment, and mixing and feeding treatment.

[0014] Preferably, the method further includes: immersing the diseased tilapia in the aqueous solution of sappanwood extract for 1 h every other day, and changing to fresh water for feeding after immersion.

[0015] Preferably, the method further includes: preparing the sappanwood extract into an aqueous solution with a certain concentration, and gavage-feeding the diseased tilapia with a gavage needle every other day.

[0016] Preferably, the method further includes: mixing sappanwood extract into the tilapia basal diet according to a certain mass fraction of the feed, and feeding normally every day.

[0017] The present invention has achieved the following technical effects compared with the prior art:

[0018] (1) The present invention firstly selects sappanwood as a therapeutic drug for tilapia bacterial meningitis, which has the effect of down-regulating the expression level of inflammatory factors in tilapia bacterial meningitis and improving the survival rate of tilapia;

[0019] (2) The Chinese herbal medicine sappanwood used in the present invention has a simple usage method and a low price, and has strong clinical transformation significance. Description of the Drawings

[0020] Figure 1 It is a graph showing the change in the mRNA expression of tight junction proteins in the co-culture model of TVEC-01 and TA-02 cells after administration of the present invention;

[0021] Figure 2 It is a chromatogram of the active ingredients of the Chinese herbal medicine used in the present invention;

[0022] Among them, Figure a is the chromatogram of brazilin, and Figure b is the chromatogram of oxybrazilin;

[0023] Figure 3 It is a graph showing the effect of the expression of related inflammatory factors in tilapia with meningitis after immersion treatment of the present invention;

[0024] Figure 4 It is a graph showing the effect of the expression of related inflammatory factors in tilapia with meningitis after gavage treatment of the present invention;

[0025] Figure 5 It is a graph showing the effect of the expression of related inflammatory factors in tilapia with meningitis after mixing and feeding treatment of the present invention;

[0026] Figure 6 It is a graph showing the pathological changes of tilapia brain tissue before and after treatment with sappanwood extract of the present invention;

[0027] Among them, Figure a is the pathological section diagram of tilapia brain tissue before treatment, Figure b is the pathological section diagram of tilapia brain tissue after treatment with 0.50 g / mL sappanwood extract by gavage, Figure c is the pathological section diagram of tilapia brain tissue after treatment with 1% sappanwood extract mixed in the basal diet, and Figure d is the pathological section diagram of tilapia brain tissue after treatment with 0.50 g / L sappanwood extract by immersion;

[0028] Figure 7 It is a graph showing the survival rate of tilapia with meningitis after treatment with an aqueous solution of sappanwood extract of the present invention;

[0029] Among them, Figure a is the survival rate graph after immersion treatment, Figure b is the survival rate graph after gavage treatment, and Figure c is the survival rate graph after mixing and feeding treatment. Specific implementation manner

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] The present invention discloses Caesalpinia sappan L. for treating tilapia bacterial meningitis. The active ingredient of Caesalpinia sappan L. is brazilin, and the brazilin is a high isoflavone compound with the molecular formula C 16 H 14 O5.

[0032] The extraction ratio of the extract of Caesalpinia sappan L. to the concentrate of Caesalpinia sappan L. is 10:1.

[0033] The LC / MS detection method for the active ingredient brazilin is as follows:

[0034] Electrospray ionization source ESI, detection in positive ion mode, scan mode selects multiple reaction monitoring MRM; mobile phase A: acetonitrile - B: 0.2% formic acid, detection time: 15 min, gradient elution 0 - 3 min, A: 35 → 10, B: 65 → 90; 6 - 6.5 min, A: 10 → 35, B: 90 → 65, flow rate 200 μL / min, injection volume 5 μL.

[0035] The present invention also discloses the application of Caesalpinia sappan L. for treating tilapia bacterial meningitis, and the application includes: having an inhibitory effect on Streptococcus agalactiae from tilapia and being able to penetrate the blood - brain barrier of tilapia.

[0036] The present invention also discloses the application method of Caesalpinia sappan L. for treating tilapia bacterial meningitis, and the method includes: immersion treatment, gavage treatment, and mixing and feeding treatment.

[0037] The application method also includes: immersing the diseased tilapia in the aqueous solution of Caesalpinia sappan L. extract for 1 h every other day, and changing to fresh water for feeding after immersion.

[0038] The application method also includes: preparing the Caesalpinia sappan L. extract into an aqueous solution with a certain concentration and gavage - feeding the diseased tilapia every other day with a gavage needle.

[0039] The application method also includes: mixing the Caesalpinia sappan L. extract into the tilapia basic feed according to a certain mass fraction of the feed and feeding normally every day.

[0040] The experimental methods used in this invention are as follows:

[0041] 1. Preparation of bacterial suspension

[0042] Under aseptic conditions, Streptococcus agalactiae ZQ0910 stored at -20°C was inoculated into BHI liquid medium at a volume ratio of 1:50, and cultured with shaking at 37°C for 6 - 8 h. The concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, and diluted 100 times with BHI liquid medium to prepare a bacterial suspension for standby.

[0043] 2. Preparation of aqueous extract of Sappanwood

[0044] Weigh 30 g of Sappanwood, soak it in 600 mL of distilled water for 2 h, then heat it to boiling (100°C) and maintain the boiling state for 60 min. Filter the solution with gauze and collect the filtrate. Decoct it again and combine the two filtrates. Evaporate and concentrate to 30 mL to obtain an aqueous extract of Sappanwood with a drug solution concentration of 1 g / mL. The aqueous extract was centrifuged at high speed (10000 r / min, 15 min), and the supernatant was filtered through a 0.22 μm microporous membrane to remove bacteria.

[0045] 3. Determination of antibacterial effect of Sappanwood

[0046] The agar diffusion method was used to measure the diameter of the antibacterial zone of Sappanwood. The two-fold dilution method was used to determine the minimum inhibitory concentration of Sappanwood.

[0047] 4. Construction of an artificial blood-brain barrier model of Nile tilapia

[0048] One night before the experiment, Matrigel was placed on ice and slowly thawed in a 4°C refrigerator. Using a pre-cooled pipette tip, Matrigel was mixed and diluted with pre-cooled serum-free L-15 medium at a ratio of 1:8. Take 50 μL of the diluted solution and evenly spread it on the surface of the Trans-well polyester fiber membrane, and place it at 37°C for 4 h until the Matrigel solidifies (the membrane turns white). Before inoculating the cells, rinse the polyester fiber membrane coated with Matrigel with complete medium. The Trans-well chamber was inoculated with astrocytes in an amount of 1×10 4 , and the lower chamber was inoculated with endothelial cells in an amount of 1×10 4 . Replace the medium 24 h after inoculating the cells.

[0049] 5. Detection of the expression level of tight junction proteins

[0050] The co - culture model of TVEC - 01 and TA - 02 cells was randomly divided into 10 groups and placed in a cell culture plate. The experimental groups were set as follows: blank group, 20 μmol / L brasilein group, 40 μmol / L brasilein group, and 80 μmol / L brasilein group. The co - culture model was taken out, the old medium was discarded, the cells were washed twice with PBS, and the dosing working solution prepared with serum - free L - 15 medium as the solvent was added. Three replicate wells were set for each group. After 4 hours, the cell culture plate was taken out, the dosing solution was discarded, the RNA of the cells was extracted, reverse transcription was carried out, and the changes in the expression levels of related tight junction proteins were detected by RT - qPCR. The primer sequences used are shown in Table 1.

[0051] 6. Analysis of the permeability of the blood - brain barrier model in vitro regulated by brasilein by high - performance liquid chromatography - tandem mass spectrometry

[0052] The standard working solution of brasilein was analyzed by LC / MS to obtain its standard curve. Considering the extremely easy oxidation property of brasilein, another standard working solution of brasilein was prepared according to this method and placed at room temperature until it was completely oxidized, and its standard curve was obtained. The completely oxidized brasilein was used as the detection object after brasilein passed through the blood - brain barrier.

[0053] The successfully constructed BBB model was selected and divided into four groups: the brasilein concentrations were 900, 450, 225 ng / mL and the blank group. There were three parallels in each group. The corresponding medicinal solutions were added to the supply pool, and the receiving pool was HBSS. 200 μL of samples were taken from the receiving pool at 15, 30, 60, 90, 120, 180, 240 min after adding the medicine, and an equal volume of blank HBSS was supplemented at the same time.

[0054] Preparation of test samples: 300 μL was aspirated into a 1.5 mL centrifuge tube, centrifuged at 8000 rmp for 10 min, the supernatant was discarded, dried by nitrogen, then 300 μL of acetonitrile was added for re - dissolution, vortexed thoroughly for 2 min, repeated twice, and filtered through a 0.22 μL needle filter membrane for LC - MS analysis.

[0055] 7. Preparation of a bacterial meningitis model in Nile tilapia

[0056] Healthy Nile tilapia were selected and injected with Streptococcus agalactiae at an injection concentration of 1.0×10 7 cfu / mL and an injection dose of 0.3 mL / 1 fish.

[0057] 8. Treatment of Nile tilapia with bacterial meningitis using extracts of Sappanwood

[0058] After tilapia was constructed with bacterial meningitis, Nile tilapia were randomly divided into four groups. There were 20 tails in each control experimental group, with 3 parallels in each group, and they were fed normally; there were 60 tails in each immersion group, which were divided into a 0.25 g / L group, a 0.35 g / L group, and a 0.50 g / L group according to the concentration of sappanwood extract during immersion. There were 3 parallels in each group, and the aqueous solution of sappanwood extract with concentrations of 0.25 g / L, 0.35 g / L, and 0.50 g / L was immersed for 1 h every other day; there were 60 tails in each gavage group, which were divided into a 0.15 g / mL group, a 0.35 g / mL group, and a 0.50 g / mL group according to the concentration of sappanwood extract during gavage. There were 3 parallels in each group, and 1 mL of the aqueous solution of sappanwood extract with concentrations of 0.15 g / mL, 0.35 g / mL, and 0.50 g / mL was gavaged every other day; there were 60 tails in each mixed feeding group, which were divided into a 1% group, a 2% group, and a 3% group according to the mass fraction of sappanwood extract added to the feed during treatment. There were 3 parallels in each group, and the feed mixed with sappanwood extract was fed every day.

[0059] 9. Detection of inflammatory factors in diseased tilapia after treatment

[0060] Sampling time: on the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days after treating tilapia with meningitis with sappanwood extract; sampling quantity: 3 tails were randomly selected from each group; sampling tissue: brain; total RNA was extracted, reverse transcribed, and the mRNA levels of related inflammatory factors in tilapia brain tissue were detected by RT-qPCR. The primer sequences used are shown in Table 2.

[0061] 10. Histopathological analysis of diseased tilapia after treatment

[0062] On the 0th and 7th days of treatment, tilapia brain tissue was taken, and after dewaxing with xylene - hydration - hematoxylin staining - differentiation - eosin staining - dehydration - clearing and mounting, it was observed under an optical microscope.

[0063] 11. Statistics of the survival rate of diseased tilapia during treatment.

[0064] Table 1:

[0065]

[0066]

[0067] Table 2:

[0068]

[0069] Example 1: Bacteriostatic effect of aqueous extract of sappanwood

[0070] Sappanwood has a good bacteriostatic effect on Streptococcus agalactiae. The diameter of the inhibition zone is 24.6 ± 0.4, and the MIC is 3.13 mg / mL.

[0071] Example 2: Effect of brazilein on the expression level of tight junction proteins

[0072] After the co - culture models of TVEC - 01 and TA - 02 cells were treated with blank medium and different concentrations of brazilein respectively, the mRNA expression changes of tight junction proteins between cells were as Figure 1 shown

[0073] Compared with the blank control group, brazilein at concentrations of 20, 40, and 80 μmol / L could significantly reduce the mRNA expression levels of JAM, ZO, and Claudin (P < 0.05).

[0074] Example 3: Optimization of chromatographic conditions for brazilein

[0075] Using acetonitrile as the mobile phase and adding 0.2% formic acid could improve the ionization efficiency of the target in positive - ion electrospray ionization (ESI), enabling better separation of the target, obtaining chromatographic peaks with sharp peaks and good symmetry, increasing the intensity of the ion peaks, and achieving good separation of brazilein and fully oxidized brazilein within 1 minute, as Figure 2 shown

[0076] The standard solutions of brazilein and fully oxidized brazilein were respectively subjected to full - scan of the first - order mass spectrometry in the positive - ion mode. It was found that the highest peak value of the full - scan response value of the brazilein standard solution (m / z = 285.1) was similar to the molecular weight of brazilein (subtracting a negative ion from the molecular weight of brazilein, 286.3). Therefore, the molecular separation peak of brazilein was determined to be 285. Then, using m / z as the parent ion of brazilein, collision energy was applied to form daughter ions, and full - scan of the second - order mass spectrometry was carried out. The daughter ion with the strongest abundance, m / z 267.0, was selected as its monitoring ion. The highest peak value of the full - scan response value of the fully oxidized brazilein standard solution (m / z = 283.1), using m / z = 283.1 as the parent ion, applying collision energy, and performing full - scan of the second - order mass spectrometry, mainly produced daughter ions such as m / z 196.0 and 265.1. According to the literature, it was judged to be oxidized brazilein, and the daughter ion with the strongest abundance, m / z 196.0, was selected as its monitoring ion.

[0077] Example 4: Permeability analysis of brazilein on the artificial blood - brain barrier model

[0078] The BBB permeability of brazilein in the 125 ng / mL group was shown in Table 3; the BBB permeability of brazilein in the 250 ng / mL group was shown in Table 4; the BBB permeability of brazilein in the 500 ng / mL group was shown in Table 5.

[0079] Table 3:

[0080]

[0081] Note: On this day, the standard curve of oxidized brazilein was y = 1.495385x + 5.088402, R 2 = 0.999.

[0082] Table 4:

[0083]

[0084] Note: On this day, the standard curve of oxidized brazilein was y = 1.495385x + 5.088402, R 2 = 0.999.

[0085] Table 5:

[0086]

[0087]

[0088] Note: On this day, the standard curve of oxidized brazilein was y = 1.495385x + 5.088402, R 2 = 0.999.

[0089] Example 5: Therapeutic effect of sappanwood extract on tilapia with bacterial meningitis

[0090] The results are as Figure 3 , Figure 4 , Figure 5 shown. After soaking, gavage, and mixing feeding, compared with the control group, the expressions of inflammatory factors TNF-α, IL-1β, IL-21, IL-10, IL-8, IL-34, and IL-12 in the tilapia brain tissue showed a decreasing trend. It shows that soaking treatment, gavage treatment, and mixing feeding treatment with sappanwood extract can reduce the expression of inflammatory factors in the brain tissue of tilapia with bacterial meningitis.

[0091] Example 6: Effect of sappanwood extract on brain tissue damage and inflammatory cell infiltration in tilapia with bacterial meningitis

[0092] The results are as Figure 6 shown. After 7 days of treatment with different treatment regimens, the brain tissue of tilapia was made into paraffin sections and stained with hematoxylin-eosin. The severity of the pathological tissue of bacterial meningitis was observed under an optical microscope and photographed for preservation. The results are as Figure 7As shown, compared with the untreated diseased tilapia, the cerebral tissue vascular congestion and exudation in the intragastric administration group of tilapia were greatly improved. The blood vessel diameter returned to near normal, the blood flow in the lumen was smooth, no obvious red blood cell aggregation and extravasation were seen, the spongy loose structure of the matrix in the cerebral parenchyma disappeared, and it returned to a tight and orderly arrangement. The cell morphology was plump, the structure was clear, the connections between nerve cells returned to normal, the width of the tissue space was moderate, and the overall tissue structure and morphological characteristics were similar to those of normal cerebral tissue. In the concomitant feeding group of tilapia, the cerebral tissue vascular congestion was alleviated, the blood flow in the lumen was smoother than before, but there was still slight red blood cell aggregation, and there was still a small amount of exudation around the blood vessel wall. The infiltration number of neutrophils decreased, but compared with the intragastric administration group, their distribution in the cerebral tissue was still relatively dense. The looseness of the matrix in the cerebral parenchyma was improved, the spongy structure was partially restored, and the tightness of cell arrangement was improved, but compared with the intragastric administration group, the connections between nerve cells were still not regular enough, and the tissue space was slightly wider. The treatment effect of the immersion group was poor, the vascular congestion and exudation were still obvious, the blood vessel diameter was dilated, a large number of red blood cells aggregated in the lumen, the infiltration of neutrophils was extensive and dense, the spongy loose structure of the matrix in the cerebral parenchyma was not significantly improved, the cell arrangement was disordered, the nerve cell morphology was blurred, the tissue space was wide, and there were significant differences in the tissue structure from normal cerebral tissue, indicating that the immersion group had a poor effect in treating tilapia bacterial meningitis.

[0093] Example 7: Therapeutic and protective effects of hematoxylin extract on tilapia with bacterial meningitis

[0094] The results are as Figure 7 shown. During the 7-day treatment process, the survival rates of the 0.25 g / L group, 0.35 g / L group, and 0.50 g / L group in the immersion treatment were 75%, 70%, and 80% respectively. The survival rates of the 0.15 g / mL group, 0.35 g / mL group, and 0.50 g / mL group in the intragastric administration treatment were 60%, 65%, and 80% respectively. The survival rates of the 1% group, 2% group, and 3% group in the concomitant feeding treatment were 65%, 55%, and 55% respectively. The survival rate of the control group was 25%. Compared with the control group, the hematoxylin extract had a therapeutic effect on bacterial meningitis.

[0095] The above are only preferred embodiments of the present invention, and do not impose any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Caesalpinia sappan L. for treating tilapia bacterial meningitis, characterized in that, The active ingredient of the Chinese herbal medicine Sappan Wood is brazilein, which is a high isoflavone compound with the molecular formula C 16 H 14 O5.

2. The Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis according to claim 1, characterized in that The extraction ratio of the extract of Caesalpinia sappan L. to Caesalpinia sappan L. is 10:

1.

3. The Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis according to claim 1, characterized in that, The LC / MS detection method for the active ingredient brazilein is as follows: Electrospray ionization source ESI, detection in positive ion mode, scan mode selects multiple reaction monitoring MRM; mobile phase A: acetonitrile - B: 0.2% formic acid, detection time: 15 min, gradient elution 0 - 3 min, A: 35 → 10, B: 65 → 90; 6 - 6.5 min, A: 10 → 35, B: 90 → 65, flow rate 200 μL / min, injection volume 5 μL.

4. Application of Chinese herbal medicine sappanwood in treating tilapia bacterial meningitis, characterized in that, The applications include: having an inhibitory effect on Streptococcus agalactiae from tilapia and being able to penetrate the blood - brain barrier of tilapia.

5. Application method of Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis, characterized in that, The methods include: immersion treatment, gavage treatment, and mixing and feeding treatment.

6. The application method of the Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis according to claim 5, characterized in that, The method further includes: immersing the diseased tilapia in the aqueous solution of Caesalpinia sappan L. extract for 1 h every other day, and feeding with fresh water after immersion.

7. The application method of the Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis according to claim 5, characterized in that, The method further includes: preparing an aqueous solution of Caesalpinia sappan L. extract at a certain concentration and gavage - feeding the diseased tilapia every other day with a gavage needle.

8. The application method of the Chinese herbal medicine sappanwood for treating tilapia bacterial meningitis according to claim 5, characterized in that, The method further includes: mixing the Caesalpinia sappan L. extract into the tilapia basal diet according to a certain mass fraction of the feeding feed and feeding normally every day.

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