Use of corilagin in the preparation of products for inhibiting the activity of isocitrate dehydrogenase / vibrio parahaemolyticus replication

Abstract

The application discloses application of corilagin in preparation of Vibrio parahaemolyticus replication inhibition products, and belongs to the field of microorganism control. It is found for the first time that corilagin can inhibit growth of Vibrio parahaemolyticus isolates from different sources, and through MIC determination, molecular docking and IDH enzyme activity detection, it is indicated that IDH is probably an important potential target point of the bacteriostatic effect of corilagin, thereby providing a new idea for development of natural source bacteriostatic agents of Vibrio parahaemolyticus.

Description

Technical Field

[0001] This invention relates to the application of Corilagin in the preparation of products that inhibit isocitrate dehydrogenase activity / Vibrio parahaemolyticus replication, and belongs to the field of microbial control. Background Technology

[0002] Vibrio parahaemolyticus ( Vibrio parahaemolyticus (This is a significant foodborne pathogen, widely found in seawater, estuaries, aquatic products, and their processing and distribution environments.) Ingestion of contaminated aquatic products can lead to gastrointestinal infections, placing ongoing pressure on aquatic product quality and safety and public health risk control.

[0003] Currently, the control of Vibrio parahaemolyticus in aquatic products mainly relies on low-temperature storage, processing sterilization, chemical disinfectants, or antimicrobial drugs. However, traditional chemical antimicrobial agents may have problems such as affecting food quality, residue risks, and increased bacterial tolerance. Naturally derived small molecule compounds have the advantages of wide availability, structural diversity, and high development potential, making them important candidate resources in the field of food microbial control.

[0004] Isocitrate dehydrogenase (IDH) is a key metabolic enzyme in the tricarboxylic acid cycle, involved in bacterial energy metabolism and reducing power generation. Inhibition of IDH activity may interfere with bacterial metabolism and energy supply, thereby affecting their growth and reproduction. Therefore, IDH can serve as a potential target for screening small antibacterial molecules against Vibrio parahaemolyticus.

[0005] Corilagin is a small polyphenol molecule of natural origin with certain biological activities. There are currently no reports on the use of corilagin for inhibiting Vibrio parahaemolyticus. Summary of the Invention

[0006] Objectives of the Invention: The first objective of this invention is to provide a novel application of corilagin in the preparation of products that inhibit isocitrate dehydrogenase activity / Vibrio parahaemolyticus replication. The second objective of this invention is to provide a drug that inhibits isocitrate dehydrogenase activity / Vibrio parahaemolyticus replication.

[0007] Technical solution: The application of Corilagin as described in this invention in the preparation of products that inhibit isocitrate dehydrogenase activity.

[0008] Furthermore, the isocitrate dehydrogenase is Vibrio parahaemolyticus isocitrate dehydrogenase.

[0009] Furthermore, the concentration of the corilagin is 0.3125~20 μM.

[0010] The application of Corilagin as described in this invention in the preparation of products that inhibit the replication of Vibrio parahaemolyticus.

[0011] Furthermore, the concentration of the corilagin is 3.125~100 μM.

[0012] Furthermore, the concentration of the corilagin is 6.25~50 μM.

[0013] The present invention discloses a drug for inhibiting isocitrate dehydrogenase activity / Vibrio parahaemolyticus replication, wherein the active substance in the drug is corilagin.

[0014] Furthermore, the drug also contains pharmaceutically acceptable carriers, solvents, or excipients.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to discover that corilagin can inhibit the growth of Vibrio parahaemolyticus isolates from different sources. By combining MIC determination, molecular docking and IDH enzyme activity detection, it is suggested that IDH may be an important potential target for corilagin to exert its antibacterial effect, providing a new idea for the development of antibacterial agents from natural sources of Vibrio parahaemolyticus. Attached Figure Description

[0016] Figure 1 The graph shows the MIC determination results of Corilagin against the laboratory-preserved Vibrio parahaemolyticus reference strain RIMD2210633 under M9G conditions. Figure 2 The MIC distribution of Corilagin against the tested strains is shown in the statistical graph. Figure 3 The diagram shows the molecular docking results between Corilagin and Vibrio parahaemolyticus IDH. Figure 4 The figure shows the effect of Corilagin on the IDH enzyme activity of Vibrio parahaemolyticus. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] Example 1: MIC determination of Corilagin against Vibrio parahaemolyticus reference strain RIMD2210633 1. Preparation of Corilagin Mother Liquor Weigh 5 mg of Corilagin powder and dissolve it completely in dimethyl sulfoxide to prepare a 20 mg / mL Corilagin stock solution. Vortex the stock solution and, if necessary, briefly sonicate or use a 37°C light-protected incubator to aid dissolution. Aliquot and store in the dark for later use.

[0019] 2. Preparation of Vibrio parahaemolyticus bacterial suspension The Vibrio parahaemolyticus isolate was inoculated into M9G medium for secondary culture until the OD600 reached approximately 1.0. 30 μL of the bacterial culture was added to 2970 μL of sterile M9G medium and mixed thoroughly to obtain a 1:100 dilution. Subsequently, 200 μL of the 1:100 dilution was added to 1800 μL of sterile M9G medium and mixed thoroughly to obtain 2 mL of a 1:1000 dilution.

[0020] The resulting 1:1000 diluted bacterial solution was used for subsequent MIC determination. 100 μL of diluted bacterial solution was added to each well and mixed with 100 μL of Corilagin working solution to make a final volume of 200 μL per well.

[0021] 3. MIC determination of Corilagin against Vibrio parahaemolyticus reference strain RIMD2210633 The minimum inhibitory concentration (MIC) of corilagin against Vibrio parahaemolyticus was determined using the 96-well plate broth microdilution method. In the preliminary screening stage, the laboratory-preserved reference strain RIMD2210633 of Vibrio parahaemolyticus was used as a representative experimental subject to evaluate the antibacterial activity of corilagin against Vibrio parahaemolyticus.

[0022] Before the experiment, the required stock solution volume was calculated based on the molecular weight of Corilagin and diluted to twice the maximum working solution of 200 μM using sterile M9G medium. Subsequently, a drug gradient was prepared in a 96-well plate using a two-fold serial dilution method, so that the drug concentrations in each well before bacterial addition were 200, 100, 50, 25, and 12.5, respectively; after adding an equal volume of bacterial solution, the final drug concentrations in each well were 100, 50, 25, 12.5, and 6.25, respectively.

[0023] A dimethyl sulfoxide (DMSO) solvent control was also set up to eliminate the influence of DMSO on the growth of the strain.

[0024] The specific procedure is as follows: Add 200 μL of 200 μM Corilagin working solution to well 1, and add 100 μL of sterile M9G medium to each of wells 2-5; add 100 μL of the solution from well 1 to well 2 and mix well, then perform serial dilutions up to well 5; after mixing well 5, aspirate and discard 100 μL, leaving 100 μL of the working solution in each of wells 1-5. Then add 100 μL of a 1:1000 diluted bacterial culture to each well, bringing the final volume of each well to 200 μL.

[0025] Each experiment included a blank control and a control group with only bacterial culture. After cultivation, the turbidity of the bacterial culture was observed visually or the OD was measured. 600 Assess bacterial growth. Use the absence of visible growth or OD (Organic Oxygen Demand) as indicators. 600 The lowest drug concentration significantly lower than that of the growth control was used as the MIC value.

[0026] The initial screening results for RIMD2210633 are as follows: Figure 1 As shown, compared with the group without Corilagin, Corilagin exhibited a visible antibacterial effect against the reference strain under M9G conditions, with a MIC value of 25 μM. The Control group consisted of the group without Corilagin, indicating that the antibacterial system was not contaminated.

[0027] Example 2: MIC determination of Corilagin on test strains from different sources Based on the 96-well plate broth microdilution method described in Example 1, 71 Vibrio parahaemolyticus isolates were further used as the main test objects, and 7 aquatic-related Gram-negative bacteria were set as controls (strains from: [1] Wang Xueying. Establishment and preliminary application of Vibrio parahaemolyticus detection system based on RPA-CRISPR / Cas12b [D]. Yangzhou University, 2025. DOI:10.27441 / d.cnki.gyzdu.2025.000641.; Zhu Qiangqiang. Study on the prevalence and spread of Vibrio parahaemolyticus in farms and markets in some areas of Jiangsu Province and phage control [D]. Yangzhou University, 2024. DOI:10.27441 / d.cnki.gyzdu.2024.001069.; Li, M. , Xu, H. , Tian, ​​Y. , Zhang, Y. , Jiao, X., & Gu, D. . (2023). Comparative (Genomic analysis reveals the potential transmission of vibrio parahaemolyticus from freshwater food to humans. Food Microbiology.) The in vitro antibacterial activity of Corilagin against test strains from different sources was evaluated. The results showed that Corilagin exhibited good in vitro antibacterial activity against most test strains, with MIC values ​​mainly distributed in the range of 6.25–12.5 μM. The results are shown in Tables 1 and 2.

[0028] Table 1. MIC determination results of Corilagin against test strains

[0029]

[0030]

[0031] Table 2. MIC distribution statistics of Corilagin against test strains

[0032] From Table 2 and Figure 2 It can be seen that the MIC values ​​of Corilagin are mainly concentrated at 6.25 μM (55 strains) and 12.5 μM (17 strains); the MIC50 is 6.25 μM and the MIC90 is 12.5 μM, indicating that Corilagin has relatively stable in vitro antibacterial activity against the test strains.

[0033] Example 3 Molecular docking of Corilagin with Vibrio parahaemolyticus IDH Molecular docking analysis was performed using Vibrio parahaemolyticus isocitrate dehydrogenase as the receptor protein and Corilagin as the ligand. Prior to docking, the receptor protein underwent dehydration, hydrogenation, and energy optimization, while the ligand was structurally optimized. Subsequently, a semi-flexible docking was performed by creating an active pocket region for the receptor, and the binding stability between the receptor and ligand was evaluated using the binding energy.

[0034] The docking results show ( Figure 3 Corilagin exhibits a good binding affinity to IDH of Vibrio parahaemolyticus, with a binding energy of -9.1 kcal / mol. Corilagin can interact with amino acid residues near the active pocket of IDH, suggesting that IDH may be an important potential target for its antibacterial activity.

[0035] Table 3. Molecular docking results between Corilagin and Vibrio parahaemolyticus IDH

[0036] Example 4: Effect of Corilagin on IDH enzyme activity of Vibrio parahaemolyticus To further verify whether corilagin affects the IDH activity of Vibrio parahaemolyticus, an IDH enzyme activity inhibition experiment was conducted. In each well of a 96-well plate, IDH (isocitrate dehydrogenase, purified through laboratory induction expression, final concentration 400 nM) and different concentrations of corilagin (final concentration range 0.3125–20 μM) were added, and the plates were incubated at 25°C for 5 minutes. A pre-mixed working solution was prepared, containing: 0.5 mM NADP⁺, 50 mM Tris-HCl (pH 7.8), 2 mM MgCl₂, 0.5 mM NADP⁺, 0.2 mg / mL BSA, and 2 mM DL-isocitrate sodium. The mixed working solution was preheated in a 37°C water bath. 950 μL of the preheated working solution was mixed with 50 μL of the incubated sample and placed in a cuvette. The reaction was carried out at 37°C for 2 minutes, and the absorbance change caused by NADPH generation at 340 nm was continuously monitored.

[0037] Depend on Figure 4 It was found that the relative IDH enzyme activity was high in the lowest concentration (0.3125 μM) Corilagin treatment group; however, the relative IDH enzyme activity gradually decreased with increasing Corilagin dosage. Specifically, the relative IDH enzyme activity in the 20 μM Corilagin treatment group was significantly lower than that in other treatment groups, indicating that Corilagin has a dose-dependent inhibitory effect on Vibrio parahaemolyticus IDH enzyme activity. Combined with in vitro MIC determination and molecular docking results, it is suggested that Corilagin may participate in the inhibition of Vibrio parahaemolyticus growth by binding to and inhibiting Vibrio parahaemolyticus IDH, thereby interfering with the bacterial tricarboxylic acid cycle and energy metabolism.

Claims

1. Application of Corilagin in the preparation of products that inhibit isocitrate dehydrogenase activity.

2. The application according to claim 1, characterized in that, The isocitrate dehydrogenase is Vibrio parahaemolyticus isocitrate dehydrogenase.

3. The application according to claim 1, characterized in that, The concentration of the kerilagin was 0.3125–20 μM.

4. Application of Corilagin in the preparation of products that inhibit the replication of Vibrio parahaemolyticus.

5. The application according to claim 4, characterized in that, The concentration of the corilagin was 3.125~100 μM.

6. The application according to claim 4, characterized in that, The concentration of the cauliflower was 6.25–50 μM.

7. A drug for inhibiting isocitrate dehydrogenase activity / Vibrio parahaemolyticus replication, characterized in that, The active ingredient in the drug is corilagin.

8. The drug according to claim 4, characterized in that, The drug also contains pharmaceutically acceptable carriers, solvents, or excipients.

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