Application of myrobalan tannic acid in preparation of medicine for neutralizing streptococcus suis hemolysin
Through the combined use of tannin with antibiotics and combined with streptococcal hemolysin, the problem that the existing technology cannot alleviate STSLS, significantly improve the treatment effect of Streptococcus suis infection, and provide new prevention and treatment drugs.
Patent Information
- Application Number
- CN202411402051.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-01-14
AI Technical Summary
Existing antibiotics are unable to effectively alleviate the poisonous shock-like syndrome (STSLS) caused by streptococcal hemolysin, so effective treatments are lacking.
Using tannin combined with antibiotics (such as amoxicillin), tannin can directly bind streptococcal hemolysin to neutralize its toxicity, thereby inhibiting hemolytic activity, and is used to prepare for prevention or treatment of streptococcal infections in swine. .
Hazilin tannin significantly improved the survival rate of mice, significantly inhibited the hemolytic activity of streptococcal hemolysin, provided new drugs to prevent and treat Streptococcus suis infection, and reduced the damage to the body by STSLS.
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Figure CN119302979A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of microbial infectious diseases and biomedicine, and particularly relates to application of terminalia chebula tannic acid in preparing a medicine for neutralizing streptococcus suis hemolysin. Background Art
[0002] Streptococcus suis is one of the most important bacterial pathogens of pigs. Streptococcus suis caused by it is a major harmful disease in the pig industry, causing huge economic losses to my country's pig industry. At the same time, Streptococcus suis is also a zoonotic pathogen distributed worldwide. In pigs and humans, Streptococcus suis can cause severe systemic invasive infections, leading to diseases such as meningitis, arthritis and sepsis, and some severely infected animals or patients will have sequelae. Studies have found that Streptococcus suis can activate NLRP3 inflammasomes through hemolysin (SLY), causing excessive production of host inflammatory factors and multiple organ failure, leading to the occurrence of toxic shock syndrome (Streptococcal toxic-shock-like syndrome, STSLS). Although commonly used antibiotics can kill Streptococcus suis, they cannot alleviate the effects of Streptococcus suis STSLS on the body. Existing studies have confirmed that suis streptolysin is the main cause of STSLS, so finding a drug that can antagonize suis streptolysin is the key to treating suis streptococcal infection. Summary of the invention
[0003] The purpose of the present invention is to provide a new application of the natural product terminalia chebula tannic acid, that is, to provide the application of terminalia chebula tannic acid in the preparation of a drug for neutralizing streptococcus suis hemolysin.
[0004] The purpose of the present invention is achieved through the following technical solutions:
[0005] The present invention found that terminalia chebula tannic acid can directly bind to streptolysin and neutralize its toxicity, and can be used in combination with other antibiotics to protect mice infected with Streptococcus suis. Based on this, the present invention provides the following applications:
[0006] Application of terminalia chebula tannic acid or its pharmaceutically acceptable salt in preparing a medicine for neutralizing streptococcic acid hemolysin.
[0007] Application of terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof in preparing a medicine for preventing or treating streptococcus suis infection.
[0008] The invention discloses a composition containing terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof and an antibiotic in the preparation of a drug for preventing or treating Streptococcus suis infection. The antibiotic includes amoxicillin.
[0009] Application of terminalia chebula tannic acid or its pharmaceutically acceptable salt in preparing medicine for neutralizing bacterial hemolysin.
[0010] Application of terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof in preparing a medicine for preventing or treating hemolysin-producing bacterial infection.
[0011] Application of a composition containing terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof and an antibiotic in the preparation of a medicine for preventing or treating hemolysin-producing bacterial infection.
[0012] The present invention also provides a drug for preventing or treating hemolysin-producing bacteria infection, which comprises terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof and an antibiotic, and may further comprise a pharmaceutically acceptable adjuvant. The hemolysin-producing bacteria include Streptococcus suis, and the antibiotic includes amoxicillin.
[0013] The present invention has the following advantages and beneficial effects:
[0014] (1) The present invention discovers the new medicinal value of terminalia chebula tannic acid and provides a new preventive and therapeutic drug for Streptococcus suis infection.
[0015] (2) Terminalia chebula tannic acid can neutralize hemolysin, the main virulence factor of Streptococcus suis, effectively inhibit its hemolytic activity, and reduce the damage of STSLS to the body.
[0016] (3) Terminalia chebula tannic acid binds to hemolysin through hydrogen bonds and hydrophobic interactions, thereby neutralizing bacterial virulence factors and exerting its effect.
[0017] (4) The combination of terminalia chebula tannic acid and amoxicillin in treating mice infected with Streptococcus suis can significantly improve the survival rate of mice. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a statistical chart of the hemolytic toxicity of terminalia chebula tannic acid to defibrinated sheep erythrocytes.
[0019] Figure 2 This is a statistical chart of the toxicity results of terminalia chebula tannic acid on mammalian Vero cells.
[0020] Figure 3 This is a statistical graph showing the hemolysis results of purified SLY protein on defibrinated sheep erythrocytes.
[0021] Figure 4 This is a statistical graph showing the inhibition of the hemolytic activity of purified SLY protein by terminalia chebula tannic acid.
[0022] Figure 5 This is the molecular docking model diagram of SLY protein and terminalia chebula tannic acid.
[0023] Figure 6This is the kinetic reaction curve of terminalia chebula tannic acid and SLY protein.
[0024] Figure 7 This is a survival curve of mice infected with Streptococcus suis treated with terminalia chebula tannic acid. DETAILED DESCRIPTION
[0025] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
[0026] The Streptococcus suis SC19 strain used in the following examples is a virulent strain isolated from the brain of a dead pig during an epidemic outbreak in Sichuan Province, China in 2005; the terminalia chebula tannic acid used is terminalia chebula tannic acid with a purity of 99.68%, purchased from MedChemExpress; the statistical analysis of the results of the examples was performed using a two-tailed unpaired t-test, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.
[0027] Example 1
[0028] The following experiment uses fresh sheep red blood cells for hemolysis experiments to detect the hemolytic toxicity of terminalia chebula tannic acid on red blood cells. Fresh sheep red blood cells were aspirated, washed with PBS, and centrifuged at 2500r / min and 4℃ for 10min before being prepared into 2% defibrinated sheep red blood cells for use. Terminalia chebula tannic acid was prepared into solutions with final concentrations of 128, 64, 32, 16, 8, and 4μg / mL, respectively. PBS and 2.5% TritonX-100 were set as negative and positive controls, respectively, and mixed with 2% defibrinated sheep red blood cells at a volume ratio of 1:1, incubated at 37℃ for 1h, centrifuged at 2500rpm for 10min, and the supernatant was aspirated into a sterile 96-well plate. The OD was measured using an enzyme reader. 543 , set up 3 biological replicates, and calculate the hemolysis rate. The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = [(OD 543 Terminalia chebula tannic acid-OD 543 Negative control) / (OD 543 2.5% Triton X-100-OD 543 negative control)] × 100%.
[0029] Results Figure 1 In the concentration range tested, terminalia chebula tannin itself could not cause hemolysis of erythrocytes.
[0030] Example 2
[0031] The cytotoxicity of terminalia chebula tannic acid on mammalian cells was tested using the CCK8 assay on Vero cells. Vero cells were inoculated in DMEM medium containing 10% FBS and cultured in an incubator at 37°C and 5% CO2. Before inoculation, the cell density was observed under a microscope. When the cells grew to 90%, the cell density was diluted to 5×10 cells / mL using DMEM medium containing 10% FBS. 4 / mL, then inoculate in a 96-well cell culture plate, add 100 μL to each well, ensure that the cells added to each well are uniform, and culture in an incubator at 37°C and 5% CO2 for 24 hours. At the same time, use DMEM containing 10% FBS to dilute terminalia chebula tannic acid, and set up 6 groups of concentration gradients, namely 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, and 4μg / mL for standby use. When the cells fill the 96-well plate, wash the cells with PBS, add fresh culture medium containing different concentrations of terminalia chebula tannic acid and continue to culture for 24 hours, replace the cell culture medium with culture medium containing 10% CCK8, and continue to culture for 2 hours and read the OD with a spectrophotometer. 450 The absorbance at 100 nm was measured, and three biological replicates were set. The calculation formula is: Cell survival rate = [(absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100%.
[0032] Results Figure 2 When the highest concentration of terminalia chebula tannic acid was 128 μg / mL, the viability of Vero cells was close to 95%, indicating that terminalia chebula tannic acid had no obvious cytotoxicity to mammalian eukaryotic cells.
[0033] Example 3
[0034] The hemolytic activity of purified suis streptococcal hemolysin (SLY) protein on erythrocytes was detected by hemolysis test. First, the purified SLY protein (0.256 mg / mL) was diluted with PBS solution at different times. The final dilution times of SLY protein were 20×, 40×, 80×, 100×, 200×, 400×, 800×, 1000×, 2000×, 4000×, and 10000×, respectively. Then, 500 μL of 2% sterile defibrinated sheep erythrocyte solution was added at a volume ratio of 1:1. A blank control (K) was set up. A 1.5 mL centrifuge tube was taken and 500 μL of 2% sterile defibrinated sheep erythrocyte solution and 500 μL of PBS solution were added thereto. Take another 1.5mL centrifuge tube, add 300μL PBS solution, 500μL 2% sterile defibrinated sheep erythrocyte solution and 200μL Triton X-100 as a positive control for complete hemolysis of erythrocytes. Place all the prepared centrifuge tubes in a 37℃ constant temperature incubator for 30min. After incubation, centrifuge at 4℃ and 2500r / min for 10min. Pipette the supernatant into a 96-well plate, 200μL per well, set three wells per tube as replicates, and use an enzyme marker to measure the absorbance value (OD543 nm ), and the hemolysis rate was calculated. The calculation formula is: cell survival rate = [(absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100%.
[0035] Results Figure 3 The SLY protein with a concentration of 0.256 mg / mL was diluted into different concentrations and incubated with red blood cells, centrifuged, and the OD value of the supernatant at 543 nm was measured by a microplate reader. The group with only red blood cells and PBS solution was set as the blank control, and the group with TritonX-100 added to cause complete rupture of red blood cells was set as the positive control. The calculation results showed that the maximum dilution factor that can achieve a 90% hemolysis rate can reach 1000 times, that is, the concentration of SLY is 0.256 μg / mL, indicating that the purified SLY protein has hemolytic activity.
[0036] Example 4
[0037] The inhibitory effect of terminalia chebula tannic acid on the hemolytic activity of purified SLY protein was evaluated by hemolysis experiment. In EP tubes, 250 μL terminalia chebula tannic acid with concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 μg / mL were mixed with 250 μL SLY protein with a final concentration of 0.256 μg / mL, and the EP tubes were placed in a 37°C constant temperature incubator for 30 minutes. After incubation, 500 μL of 2% sterile defibrinated sheep erythrocyte solution was added to each EP tube. An EP tube with 250 μL purified SLY protein, 250 μL PBS solution and 500 μL red blood cell solution was set up as a control without drug addition (0); an EP tube with 500 μL PBS solution and 500 μL red blood cell solution was set up as a blank control (K); an EP tube with 300 μL PBS solution, 500 μL red blood cell solution and 200 μL Triton X-100 was set up as a control for complete hemolysis (T). The above EP tubes were placed in a 37°C constant temperature incubator for incubation for 30 minutes, and then centrifuged at 2500 r / min for 10 minutes, and the supernatant of each well was aspirated into a new 96-well plate, 200 μL per well. Three replicates were set up. The absorbance value (OD 543nm ), collect data and analyze the inhibitory effect of terminalia chebula tannic acid on the activity of streptococcal hemolysin of suis.
[0038] Results Figure 4 , under the action of terminalia tannic acid with a concentration higher than 0.5μg / mL, the hemolysis rate of erythrocytes was lower than 2%; when the terminalia tannic acid concentration was higher than 0.25μg / mL, the difference was extremely significant compared with the group with only SLY protein and erythrocytes added (P<0.001); the visual observation graph showed that when the terminalia tannic acid concentration was higher than 2μg / mL, no obvious hemolysis was observed, and terminalia tannic acid had a good effect of inhibiting SLY protein hemolysis in all the concentration ranges used. These results show that terminalia tannic acid has a good effect of inhibiting SLY protein activity in vitro and has potential value for further drug research.
[0039] Example 5
[0040] The molecular docking mode of SLY and terminalia chebula tannic acid was analyzed using Cb-dock2. The crystal structure of SLY is from the Protein Data Bank (PDB) http: / / www.rcsb.org / structure / 3HVN. The docking method adopts ATUO blindDocking, and the parameters are set to default. Among the five docking conformations generated, the one with the best affinity was selected for analysis, with a Vina score of -16.9. The results are shown in Figure 5In this docking conformation, 20 amino acids including E53, Y54, D55, N57, P58, E377, Y378, I379, T381, V212, V211, D209, F208, Y184, S185, M186, S187, and E198 form a good hydrophobic binding pocket cavity, and the terminalia tannic acid binds tightly to the binding pocket of the SLY protein and forms a key hydrogen bond interaction. The results show that terminalia tannic acid can directly bind to the SLY protein and inhibit its hemolytic activity.
[0041] Example 6
[0042] The equilibrium dissociation constants of SLY and chebula tannic acid were determined by isothermal titration calorimetry (ITC) technology, further proving the interaction between SLY protein and chebula tannic acid. First, the SLY protein and chebula tannic acid were degassed, and the nano ITC software was opened for preheating. The instrument was cleaned with 300mL of Decon lotion, ultrapure water and PBS buffer respectively. The instrument setting parameters were as follows: temperature 25℃, number of sample injections 20, 2.5μL per injection. 300μL of degassed 0.05mmol / L SLY protein was drawn with an injection needle and slowly added to the reaction pool to avoid the generation of bubbles. At the same time, 300μL of ultrapure water was added to the reference pool as a control. 50μL of 0.5mmol / L chebula tannic acid, the sample to be tested, was drawn with a titration needle, and titrated after the instrument was balanced. The titration curve was analyzed with Nano Analyzer, and the binding force and binding curve were analyzed with the Independent model.
[0043] Results Figure 6 The equilibrium binding constant (KD) of terminalia chebula tannic acid and SLY protein was 8.03 μM, and the stoichiometric ratio was 2.854:1, indicating that there was a strong binding force between the two. In addition, the thermodynamic parameters in the ITC experiment showed that the binding enthalpy (△H) and entropy change (△S) were both negative, indicating that hydrogen bonds and van der Waals forces were the interaction forces between terminalia chebula tannic acid and hemolysin protein.
[0044] Example 7
[0045] The mice used in the following experiments were 7-week-old female SPF-grade BALB / c mice purchased from Huazhong Agricultural University. The animal experiments complied with animal ethical requirements, and all experiments were conducted under the guidance of the Animal Experiment Protection and Supervision Management Committee of Huazhong Agricultural University (HZAUMO-2021-0008).
[0046] The mice were divided into 5 groups (10 mice in each group), namely SC19 control group, amoxicillin treatment group, terminalia chebula tannic acid treatment group, combined treatment group and blank control group. The overnight cultured SC19 were transferred into TSB medium containing 5% fetal bovine serum at a volume ratio of 1:100 and cultured at 37°C until OD 600 =0.6. After centrifugation at 4°C and 10000 rpm for 10 min, the cells were collected and suspended in PBS (pH 7.4). Mice in the blank control group were intraperitoneally injected with 200 μL PBS as a control, and mice in the other four groups were intraperitoneally injected with 200 μL of 1.25×10 9 CFU / mL of Streptococcus suis SC19. Six hours after infection, mice were treated by intraperitoneal injection of chebula tannic acid (10 mg / kg) and amoxicillin (5 mg / kg), chebula tannic acid (10 mg / kg) alone, and amoxicillin (5 mg / kg) alone. The SC19 control group and the blank control group were injected with the same dose of PBS, and the mice were observed for 7 days and the survival curve was plotted.
[0047] Results Figure 7 , all mice in the untreated group died 2 days after infection, while the survival rate of infected mice in the combined treatment group of terminalia chebula tannic acid and amoxicillin reached 90%. Compared with the survival rate of the groups treated with antibiotics amoxicillin and terminalia chebula tannic acid alone, the protection rate of infected mice in the combined group increased by 40% and 70%, respectively. This shows that terminalia chebula tannic acid combined with clinical antibiotics has a better protective effect on mice infected with Streptococcus suis.
[0048] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. Use of terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for neutralizing streptococcal hemolysin.
2. Use of terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating Streptococcus suis infection.
3. Use of a composition containing terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof and an antibiotic in the preparation of a medicament for preventing or treating Streptococcus suis infection.
4. The use according to claim 3, characterized in that: The antibiotics include amoxicillin.
5. Use of terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for neutralizing bacterial hemolysin.
6. Use of terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating infection by hemolysin-producing bacteria.
7. Use of a composition containing terminalia chebula tannic acid or a pharmaceutically acceptable salt thereof and an antibiotic in the preparation of a medicament for preventing or treating infection by hemolysin-producing bacteria.
8. A drug for preventing or treating infection by hemolysin-producing bacteria, characterized in that: The invention comprises myrobalan tannic acid or a pharmaceutically acceptable salt thereof and an antibiotic.
9. The drug according to claim 8, characterized in that: Also contains pharmaceutically acceptable excipients.
10. The drug according to claim 8, characterized in that: The hemolysin-producing bacteria include Streptococcus suis, and the antibiotic includes amoxicillin.