Application of EGCG (epigallocatechin gallate) and preparation thereof in inhibiting vibrio vulnificus or diseases caused by vibrio vulnificus

Abstract

The invention relates to the technical field of biological medicines, in particular to application of EGCG (epigallocatechin gallate) and a preparation thereof in inhibiting vibrio vulnificus or diseases caused by the vibrio vulnificus. It is found for the first time that EGCG can inhibit growth of vibrio vulnificus, destroy cell membranes of the vibrio vulnificus and inhibit formation of mycoderm, meanwhile, TLR4, MyD88 and / or NF-kappa B signal channels can be adjusted, host inflammatory response and organ dysfunction are relieved, and therefore EGCG can be used for preventing and / or treating vibrio vulnificus sepsis caused by the vibrio vulnificus; the blank of the existing treatment means in the aspect of anti-vibrio vulnificus and immune double-target intervention is filled, and important theoretical significance and application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of EGCG and preparations thereof in inhibiting Vibrio vulnificus or diseases caused by Vibrio vulnificus. Background Art

[0002] Vibrio vulnificus is a halophilic, Gram-negative bacterium widely found in marine and estuarine environments. It can enter the human body through ingestion of contaminated aquatic products or open wounds. It is an opportunistic pathogen with a high mortality rate. Infection with Vibrio vulnificus can cause sepsis. Immune imbalance, particularly abnormal macrophage polarization, is a key component of the pathogenesis of sepsis caused by Vibrio vulnificus infection.

[0003] Currently, clinical treatment for Vibrio vulnificus infection is still primarily based on antibiotics, but their efficacy is limited due to factors such as rapid proliferation of the pathogen, biofilm protection, and the effects of virulence factors. Furthermore, the immune imbalance and "cytokine storm" induced during infection are difficult to control with a single antimicrobial drug. While immunosuppressants can alleviate inflammation to a certain extent, they may lead to secondary infections or further decline in immune function. Therefore, the development of treatments that combine antimicrobial and immunomodulatory functions is a significant unmet clinical need. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the use of EGCG and its preparations in inhibiting Vibrio vulnificus or diseases caused by Vibrio vulnificus.

[0005] The present invention provides the use of epigallocatechin gallate (EGCG) or an epigallocatechin gallate preparation in preparing a product for inhibiting Vibrio vulnificus.

[0006] Furthermore, the EGCG concentration is 1.5 μg / mL to 100 μg / mL. The concentration is the antibacterial concentration used, and the MIC value of EGCG against Vibrio vulnificus is 12.5 μg / mL. The MIC value refers to the minimum inhibitory concentration (MIC), which is the lowest concentration of an antibacterial drug that can completely inhibit the growth of a certain bacteria in an in vitro test.

[0007] The present invention screened 13 commonly used Vibrio vulnificus inhibitory drugs and found that in addition to antibiotics, EGCG has the best anti-Vibrio vulnificus effect and shows the prospect of being used in combination with antibiotics.

[0008] The present invention provides the use of epigallocatechin gallate (EGCG) or an epigallocatechin gallate preparation in preparing a product for preventing and / or treating diseases caused by Vibrio vulnificus.

[0009] Furthermore, the disease caused by Vibrio vulnificus includes Vibrio vulnificus sepsis.

[0010] The dosage of EGCG is 5-20 mg / kg, specifically 10 mg / kg.

[0011] The prevention and / or treatment of diseases caused by Vibrio vulnificus include:

[0012] (1) inhibiting the growth of Vibrio vulnificus; and / or

[0013] (2) destroying the bacterial membrane of Vibrio vulnificus; and / or

[0014] (3) inhibiting Vibrio vulnificus biofilm formation; and / or

[0015] (4) inhibiting the upregulation of TLR4 pathway-related proteins induced by Vibrio vulnificus stimulation; and / or

[0016] (5) inhibiting the upregulation of MyD88 pathway-related proteins induced by Vibrio vulnificus stimulation; and / or

[0017] (6) Inhibit the upregulation of NF-κB pathway-related protein expression induced by Vibrio vulnificus stimulation.

[0018] In the application of the present invention, the EGCG preparation includes EGCG and pharmaceutically acceptable excipients.

[0019] Furthermore, the pharmaceutically acceptable excipients include at least one or a combination of two or more of solvents, solubilizers, co-solvents, emulsifiers, disintegrants, stabilizers, plasticizers, penetration enhancers and / or sustained-release agents.

[0020] Furthermore, the solvent includes water or a buffer solution;

[0021] The solubilizing agent includes a surfactant and / or a cyclodextrin;

[0022] The cosolvent includes organic acids and sodium salts thereof, amide compounds, inorganic salts and / or cyclodextrin and its derivatives;

[0023] The emulsifier includes surfactants, polysaccharides, phospholipids, proteins and / or polyoxyethylene fatty alcohol ethers;

[0024] The disintegrants include starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and microcrystalline cellulose;

[0025] The stabilizer includes antioxidants, chelating agents, cyclodextrin and its derivatives and / or surfactants;

[0026] The plasticizer includes polyols, organic esters and / or natural polymer derivatives;

[0027] The penetration enhancer includes alcohol compounds, terpene compounds and / or fatty acid esters;

[0028] The sustained-release agent includes hydrophilic gel, ethyl cellulose, polyethylene, polyvinyl chloride, ethylene-vinyl acetate copolymer and / or polymethacrylate.

[0029] In the application of the present invention, the dosage form of the EGCG preparation includes an oral dosage form and / or an injection dosage form.

[0030] Furthermore, the oral dosage forms include: suspensions, emulsions, powders, granules, tablets and / or capsules;

[0031] The injection dosage forms include: solution injection, suspension injection and / or emulsion injection.

[0032] In the application of the present invention, the product includes a drug and / or a drug combination.

[0033] The present invention discovered for the first time that EGCG can inhibit the growth of Vibrio vulnificus, destroy the cell membrane of Vibrio vulnificus and inhibit the formation of biofilm, and at the same time regulate TLR4, MyD88 and / or NF-κB signaling pathways, alleviate host inflammatory response and organ dysfunction, and thus can be used to prevent and / or treat Vibrio vulnificus sepsis caused by Vibrio vulnificus; filling the gap in existing treatment methods in the dual-target intervention of anti-Vibrio vulnificus and immunity, and has important theoretical significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is the structural formula of EGCG;

[0035] Figure 2 Shown is the growth curve of Vibrio vulnificus ATCC27562 under the action of different concentrations of EGCG;

[0036] Figure 3 Shown is the survival rate of Vibrio vulnificus ATCC27562 under the action of different concentrations of EGCG;

[0037] Figure 4 The survival rates of 6 clinical Vibrio vulnificus strains under the action of different concentrations of EGCG (0, 12.5, 25, 50 μg / mL) are shown;

[0038] Figure 5 The results show the inhibitory effect of EGCG on the biofilm formation of Vibrio vulnificus ATCC27562;

[0039] Figure 6The results show the destructive effect of EGCG on the biofilm of Vibrio vulnificus ATCC27562;

[0040] Figure 7 Shows the cytotoxicity of EGCG on BMDMs;

[0041] Figure 8 The bacterial loads in peritoneal lavage fluid, spleen and liver of mice with Vibrio vulnificus sepsis are shown;

[0042] Figure 9 Shows the expression of target proteins in each group after RAW264.7 cells were treated. DETAILED DESCRIPTION

[0043] The present invention provides the use of EGCG and its preparations in inhibiting Vibrio vulnificus or diseases caused by Vibrio vulnificus. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] The use of the terms "including," "having," or "comprising," including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context. The use of any and all examples or exemplary language herein, such as "for example," or "including," is intended merely to better illustrate the present application and does not limit the scope of the present application unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the present application.

[0045] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0046] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0047] The terms used in the embodiments of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" describes the association relationship between associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " in "and / or" indicates that the associated objects are in an "and" relationship.

[0048] EGCG, epigallocatechin gallate, derived from commercially available powder, CAS No.: 989-51-5; molecular formula: C 22 H 18 O 11 ;

[0049] Dissolve 10 mg of EGCG in 1 mL of DMSO to prepare a 10 mg / mL EGCG stock solution for later use.

[0050] Clinical strain ATCC27562 of Vibrio vulnificus and six strains of Vibrio vulnificus isolated from the blood of patients with sepsis were cultured in LB medium containing 2% sodium chloride at 30°C.

[0051] The present invention has the following advantages:

[0052] (1) Novel uses: It was discovered for the first time that EGCG can be used to treat Vibrio vulnificus infection and its resulting sepsis;

[0053] (2) Multiple mechanisms of action: It can directly inhibit bacteria and regulate immunity, with clear mechanisms of action;

[0054] (3) Good biosafety: Based on natural products, it has low toxicity and side effects;

[0055] (4) Multi-application potential: can be expanded to the treatment of other Gram-negative bacterial infections;

[0056] (5) Strong technical feasibility: Animal experiments have verified its efficacy and it has good prospects for industrialization.

[0057] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0058] Example 1 Screening of common antimicrobial drugs that inhibit Vibrio vulnificus

[0059] Vibrio vulnificus ATCC27562 was inoculated onto Columbia blood agar plates and then incubated in a 37°C incubator for 18 hours. According to the operating procedures of the VITEK 2 fully automatic microbial analyzer, single colonies were selected from the blood plates during the logarithmic growth phase and sterile saline was added to adjust the bacterial suspension to a McFarland turbidity of 0.5. The GN drug susceptibility test card was placed in a VITEK filler, filled with liquid, and then placed in a reader for reading. This revealed the susceptibility of the tested strain to various clinical antimicrobial drugs. Compared to single drugs such as baicalin and resveratrol, EGCG has a lower MIC value. Besides antibiotics, EGCG has the best antibacterial effect against Vibrio vulnificus and shows promise for combined use with antibiotics.

[0060] Table 1. MIC values ​​of common drugs against Vibrio vulnificus

[0061] Common antibacterial drugs MIC value Cefazolin 16 μg / mL Amikacin 8 μg / mL Co-trimoxazole ≤20 μg / mL Piperacillin / tazobactam ≤4 μg / mL Meropenem ≤2 μg / mL Terminalia chebula water extract 3.9 mg / mL Baicalin 45 μg / mL Resveratrol 125 μg / mL Artesunate ≤120 μg / mL Puerarin 150 μg / mL Glabridin 25 μg / mL EGCG 12.5 μg / mL

[0062] Example 2 In vitro experiment on the inhibitory effect of EGCG on the growth of Vibrio vulnificus

[0063] In order to evaluate the Figure 1 To investigate the antibacterial efficacy of EGCG (shown in Figure 2), Vibrio vulnificus ATCC27562 was incubated with varying concentrations of EGCG (0–200 μg / mL) at 30°C and 220 rpm shaking. A control group was treated with blank LB medium. The absorbance at 600 nm was measured at predetermined time points, and bacterial growth curves were plotted. When the bacteria reached the plateau phase, the culture medium was collected, and bacterial viability was calculated according to the following formula:

[0064] Bacterial activity (%) = (As-As') / (Ac-Ac') × 100%

[0065] Wherein, As and Ac represent the absorbance values ​​of the EGCG-treated group and the control group, respectively, while As' and Ac' represent the baseline absorbance values ​​when EGCG was administered.

[0066] The results showed that EGCG inhibited bacterial growth in a dose-dependent manner, and the bacterial survival rate decreased with the increase of EGCG concentration ( Figure 2 、 3 ).

[0067] Subsequently, six clinically isolated Vibrio vulnificus strains (Vibrio vulnificus 8000, Vibrio vulnificus 9211, Vibrio vulnificus 6317, Vibrio vulnificus 6239, Vibrio vulnificus 3632, and Vibrio vulnificus 5113) were co-incubated with different concentrations of EGCG (0, 12.5, 25, and 50 μg / mL) to evaluate the inhibitory effect of EGCG on clinical strains. The results showed that EGCG showed a significant inhibitory effect on different clinical strains, and the bacterial survival rate decreased with increasing EGCG concentration ( Figure 4 ).

[0068] Example 3 Observation of EGCG's ability to destroy bacterial membranes and form biofilms

[0069] The anti-biofilm activity of EGCG was evaluated using crystal violet staining. To evaluate its effect on biofilm formation, Vibrio vulnificus was incubated with different concentrations of EGCG (0-100 μg / mL). 200 μL of bacterial suspension was added to each well of a 96-well plate and incubated at 37°C for 24 hours. To evaluate the destructive effect of EGCG on pre-formed biofilms, Vibrio vulnificus was first cultured in LB medium in a 96-well plate at 37°C for 24 hours to allow biofilm formation. Subsequently, non-adherent bacterial cells and residual LB medium were removed by washing with PBS. The pre-formed biofilms were then exposed to EGCG (0-100 μg / mL) for 24 hours. In both experiments, the resulting biofilms were washed with PBS, stained with crystal violet, and observed under an inverted microscope. The biofilms stained with crystal violet were then dissolved with ethanol, and the biofilm mass was quantified by measuring OD595. The anti-biofilm efficiency was calculated as follows:

[0070] Biofilm inhibition (%) = (1-As / Ac) × 100%;

[0071] Biofilm destruction rate (%) = (1-As / Ac) × 100%;

[0072] Wherein, As and Ac represent the absorbance values ​​of the EGCG-treated group and the control group, respectively.

[0073] The results showed that EGCG inhibited the formation of Vibrio vulnificus biofilm in a dose-dependent manner. Similarly, it had a destructive effect on the already formed biofilm and significantly reduced the amount of biofilm ( Figure 5 and 6 )

[0074] Example 4 EGCG Cell Safety Evaluation

[0075] To evaluate the cytotoxicity of EGCG, bone marrow-derived macrophages (BMDMs) were seeded in 96-well plates. When the cell density reached 70-80%, the cells were exposed to various concentrations of EGCG (0-100 μg / mL) and incubated for 24 hours. The culture medium was then replaced with fresh culture medium containing 10% CCK-8 reagent, and the cells were incubated for a further 2 hours. The absorbance of each well at 450 nm was measured, and cell viability was calculated.

[0076] The results showed that EGCG had almost no cytotoxicity to BMDM within the concentration range of 0-100 μg / mL, indicating that EGCG has good biocompatibility ( Figure 7 ).

[0077] Example 5 Construction of Vibrio vulnificus sepsis model in mice and the effect of EGCG intervention

[0078] To establish a Vibrio vulnificus sepsis mouse model, mice were intraperitoneally inoculated with 1×10 7 Colony-forming units (CFU) of Vibrio vulnificus were detected in the mice, and EGCG (10 mg / kg) was injected intravenously 1 hour and 3 hours after inoculation. The control mice were injected intraperitoneally with 0.1 mL of PBS. 12 hours after inoculation, the mice were euthanized, and the peritoneal lavage fluid and major organs were collected for further analysis. The spleen and liver were homogenized, and the peritoneal lavage fluid and tissue homogenate were taken for bacterial smearing. In the control group and the EGCG-treated group, there was no bacterial growth in the abdominal cavity, spleen, and liver of the mice; a large number of bacteria were observed in the abdominal cavity, spleen, and liver of the mice with Vibrio vulnificus sepsis; in the mice with Vibrio vulnificus sepsis + EGCG-treated group, bacterial smearing in the abdominal cavity, spleen, and liver showed a decrease in bacterial load, indicating that EGCG inhibited the spread and infection of Vibrio vulnificus in mice ( Figure 8 ).

[0079] Example 6 EGCG regulation experiment on TLR4 / MyD88 / NF-κB pathway protein expression

[0080] RAW264.7 cells were cultured in 6-well plates and treated with EGCG (12.5 μg / mL) and Vibrio vulnificus (infection coefficient = 10) for 90 minutes. The cells were lysed, and the proteins were extracted and quantified. Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed, and the membrane was transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking, the membrane was incubated with the primary antibody at 4°C overnight, and then incubated with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. A chemiluminescence imaging system was used to display protein bands and quantify target protein expression. There was almost no change in the protein expression of RAW264.7 cells in the control group and the EGCG-treated group alone. Vibrio vulnificus stimulation induced upregulation of the expression of proteins related to the TLR4 / MyD88 / NF-κB pathway. Treatment with EGCG inhibited the activation of proteins in this pathway ( Figure 9 ).

[0081] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of epigallocatechin gallate or an epigallocatechin gallate preparation in the preparation of a product for inhibiting Vibrio vulnificus.

2. The use according to claim 1, characterized in that The concentration of the epigallocatechin gallate is 1.5 μg / mL to 100 μg / mL.

3. Use of epigallocatechin gallate or an epigallocatechin gallate preparation in the preparation of a product for preventing and / or treating diseases caused by Vibrio vulnificus.

4. The use according to claim 3, characterized in that The diseases caused by Vibrio vulnificus include Vibrio vulnificus sepsis.

5. The use according to claim 3, characterized in that The dosage of the epigallocatechin gallate is 5 to 20 mg / kg.

6. The use according to claim 3, characterized in that The prevention and / or treatment of diseases caused by Vibrio vulnificus include: (1) inhibiting the growth of Vibrio vulnificus; and / or (2) destroying the bacterial membrane of Vibrio vulnificus; and / or (3) inhibiting Vibrio vulnificus biofilm formation; and / or (4) inhibiting the upregulation of TLR4 pathway-related proteins induced by Vibrio vulnificus stimulation; and / or (5) inhibiting the upregulation of MyD88 pathway-related proteins induced by Vibrio vulnificus stimulation; and / or (6) Inhibit the upregulation of NF-κB pathway-related protein expression induced by Vibrio vulnificus stimulation.

7. The use according to claim 1 or 3, characterized in that The epigallocatechin gallate preparation comprises EGCG and pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The pharmaceutically acceptable excipients include at least one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, disintegrants, stabilizers, plasticizers, penetration enhancers and / or sustained-release agents.

9. The use according to claim 1 or 3, characterized in that The dosage form of the epigallocatechin gallate preparation includes an oral dosage form and / or an injection dosage form.

10. The use according to claim 1 or 3, characterized in that: The products include drugs and / or drug combinations.

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