A method for inhibiting aflatoxin production using benzoic acid derivatives
By using the benzoic acid derivative 2-(3-chlorobenzoamido)-5-{4-[2-oxo-2-(pyrrolidone-1-yl)ethyl]piperazin-1-yl}benzoic acid, the problem of aflatoxin production was solved, and effective inhibition of AFB1 and AFB2 was achieved, thus improving food safety.
Patent Information
- Application Number
- CN202311409133.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-26
- Publication Date
- 2025-11-28
- Estimated Expiration
- 2043-10-26
AI Technical Summary
Existing technologies are insufficient to effectively inhibit the production of aflatoxins, especially AFB1 and AFB2, which pose a serious threat to food safety and health.
The benzoic acid derivative 2-(3-chlorobenzoamido)-5-{4-[2-oxo-2-(pyrrolidone-1-yl)ethyl]piperazin-1-yl}benzoic acid at a concentration of 6.8 μM-68 μM was used to inhibit the synthesis of toxins by Aspergillus flavus.
It effectively inhibits the production of aflatoxin, especially at a concentration of 68 μM, significantly reducing the accumulation of aflatoxin in food and ensuring food safety.
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Figure CN117652611B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of aflatoxin prevention and control, and particularly relates to a method for inhibiting aflatoxin production by using benzoic acid derivatives. BACKGROUND
[0002] A benzoic acid derivative, 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid, is a complex structure with multiple functional groups of 3-chlorobenzamido, pyrrolidine ring, piperazine ring and benzoic acid.
[0003] The molecular formula of 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid is C24H27ClN4O4, and the molecular weight is 470.9. The SMILES code is C1CCN(C1)C(=O)CN2CCN(CC2)C3=CC(=C(C=C3)NC(=O)C4=CC(=CC=C4)Cl)C(=O)O; the structural formula is:
[0004]
[0005] Aflatoxins are highly toxic and carcinogenic secondary metabolites produced mainly by Aspergillus flavus and A. parasiticus. During crop growth, harvesting, storage and processing, these molds can contaminate major food crops such as corn, peanuts and nuts when the environmental temperature and humidity are suitable. The main types of aflatoxins include AFB1, AFB2, AFG1 and AFG2, among which AFB1 is the most common and toxic form, which is classified as a class 1 carcinogen by the International Agency for Research on Cancer. In addition to its carcinogenicity, ingestion of food contaminated with aflatoxins can also cause various diseases such as malnutrition, infertility, endocrine problems, congenital fetal malformations and immune deficiency, resulting in serious economic and health impacts. Therefore, inhibiting the accumulation of aflatoxins is crucial for protecting food safety and people's health. SUMMARY
[0006] The present application provides a method for inhibiting aflatoxin production by using benzoic acid derivatives.
[0007] Preferably, the benzoic acid derivative is 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid.
[0008] Preferably, the concentration of the 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid is 6.8 μM to 68 μM.
[0009] Preferably, the concentration of the 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid is 68 μM.
[0010] Preferably, the aflatoxin is AFB1 or AFB2.
[0011] Preferably, the aflatoxin is produced by Aspergillus flavus.
[0012] The present application also provides a preparation for inhibiting the production of aflatoxin, wherein the active ingredient comprises 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid.
[0013] Preferably, the concentration of the 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid is 6.8 μM to 68 μM.
[0014] The present application also provides the use of 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid, or the method or preparation as described above, in inhibiting the production of aflatoxin.
[0015] Preferably, the concentration of the 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid is 68 μM, the aflatoxin is AFB1 or AFB2, and the aflatoxin is produced by Aspergillus flavus.
[0016] Advantages of the present application:
[0017] 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid can effectively inhibit the synthesis of aflatoxin by Aspergillus flavus, and the inhibitory effect is best when the concentration is 68 μM. It is of great significance for the prevention and control of aflatoxin and the protection of food safety. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 is the relative fluorescence intensity of aflatoxins.
[0019] Figure 2 is the relative fluorescence intensity of aflatoxins. DETAILED DESCRIPTION
[0020] The experimental methods used in the following examples are conventional methods unless otherwise specified.
[0021] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.
[0022] The following examples are further illustrations of the application and are not intended to limit the same.
[0023] Example 1: 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid inhibits aflatoxin production
[0024] Aspergillus flavus (NRRL 3357) was obtained from the United States Department of Agriculture-Agricultural Research Service (USDA-ARS); starter culture of the strain: a small amount of the strain stored in glycerol at -80°C was uniformly spread on PDA medium and incubated at 37°C in a constant temperature incubator with aeration for 4 days, and green spores were observed to be produced.
[0025] The PDA medium was prepared as follows: 200 g of potatoes were washed, peeled, and cut into small pieces, and boiled until soft (boiled for 30 minutes, until the pieces could be broken by a glass rod), filtered through four layers of gauze, and then 20 g of glucose and 15 g of agar were added, and the mixture was heated and stirred until uniform, and then the volume was made up to 1000 ml with water, and the mixture was sealed with a breathable film, autoclaved at 121°C for 15 minutes, and then removed and stored in a 4°C refrigerator until use.
[0026] Aspergillus flavus spore counting: (turbidimetry) the spores of Aspergillus flavus were repeatedly washed with 0.05% Tween 20 water, and the spores were finally dispersed into particles, and then diluted by a factor of two, and a suitable amount of the spore suspension sample was loaded onto a cell counting plate, and the number of spores was calculated by an automatic cell counter (Bio-rad), and a spore suspension with an initial density of 0.8 x 106 spores / ml was obtained by dilution with Tween 20 (0.05%) water, and the suspension was prepared on the same day and used on the same day.
[0027] 1 liter of GMS liquid synthetic medium was prepared as follows: 50 g of glucose, 3 g of (NH4)2SO4, 2 g of MgSO4-7H2O, 10 g of KH2PO4, 700 mg of Na2B4O7-10H2O, 680 mg of NaMoO4-2H2O, 10 mg of FeSO4-7H2O, 300 mg of CuSO4-5H2O, 1.76 g of ZnSO4-7H2O and 110 mg of MnSO4-H2O were dissolved in water, and water was added to make up to 1 liter. After sterilization by autoclaving (121 °C) for 15 minutes, the GMS liquid synthetic medium was obtained.
[0028] Preparation of 2-(3-chlorobenzamide)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid stock solution: 5 mg of 2-(3-chlorobenzamide)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid powder (purchased from Shanghai Tauto Biotech Co., Ltd., product catalog number F687-0983) was dissolved in 0.663 mL of pure DMSO, and after sterilization by filtration through a 0.22 μm filter, a 16 mM compound stock solution was obtained.
[0029] Aspergillus flavus 2-(3-chlorobenzamide)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid exogenous experiment: 18 mL of GMS liquid synthetic medium, 2 mL of Aspergillus flavus spore suspension, and 85 μL of compound stock solution + 15 μL of DMSO (final compound concentration 68 μM), 8.5 μL of compound stock solution + 91.5 μL of DMSO (final compound concentration 6.8 μM), or only 100 μL of DMSO, or only 100 μL of GMS liquid synthetic medium were added to each flask as controls, and the flasks were incubated in a 28 °C, 180 rpm shaker for 6 days. 200 μL of culture medium was taken from each flask on the 2nd to 6th day for aflatoxin extraction, and thin layer chromatography (TLC) was used for detection.
[0030] TLC method for detection of aflatoxins: Mark the rectangular chromatography plate, one complete plate can be used for 19 samples. Draw a straight line on the lower edge of the glass plate with a pencil (the position where the liquid diffusion mark reaches and the sample position line); choose the smooth side of the glass plate as the sample line and mark it with a pencil, draw a dot every 1 cm as the sample position mark. Prepare the developing agent according to the volume ratio of chloroform to acetone 9:1 (9 mL of chloroform and 1 mL of acetone), 5 mL of developing agent is needed for one glass plate. Take 100 μL of the bacterial solution with a syringe and add it to a 1.5 mL centrifuge tube, add 50 μL of chloroform for extraction, mix thoroughly with a vortex mixer, centrifuge at 12000 rpm for 6 minutes, take 10 μL of the lower layer (chloroform layer) with a syringe, vertically touch the sample mark position (cross), and slowly discharge the liquid. The sample concentration is 2 μg / mL, and 10 μL is used for sample. Add 1 mL of developing agent to the glass developing tank to rinse the groove, discard the remaining developing agent, and then add 5 mL of developing agent again. Place the glass plate vertically in the liquid with both hands, contact the bottom of the groove, and then gently release your hands. Place the glass plate obliquely on the side of the developing tank, and after about 4 minutes, take out the glass plate and take a photo using a gel imager (Bio-rad).
[0031] TLC images of the change in aflatoxin content over time in Aspergillus flavus culture medium with different concentrations of 2-(3-chlorobenzamide)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid are shown in Figure 1 The synthesis of aflatoxins after 2 days, 3 days, 4 days, 5 days, and 6 days of treatment with 68 μM of 2-(3-chlorobenzamide)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid was significantly inhibited.
[0032] The relative fluorescence intensity quantification of the TLC images of the change in aflatoxin content over time in Aspergillus flavus culture medium with different concentrations of 2-(3-chlorobenzamide)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid is shown in Figure 2As shown, the fluorescence intensity of aflatoxins in the samples treated with 68 μM of 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid after 2, 3, 4, 5 and 6 days of incubation was significantly lower than that of the control samples with 5% DMSO, indicating that the synthesis of the toxin was significantly inhibited. The samples treated with 6.8 μM of 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid showed some inhibition of aflatoxins, indicating that the inhibition of aflatoxins was more obvious with the increase of the concentration of 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid.
[0033] As can be seen from the above experiment, 2-(3-chlorobenzamido)-5-{4-[2-oxo-2-(pyrrolidin-1-yl)ethyl]piperazin-1-yl}benzoic acid can effectively inhibit the synthesis of aflatoxins.
Claims
1. A method for inhibiting aflatoxin production for non-disease diagnosis and treatment purposes, characterized in that, The aflatoxin is derived from 2-(3-chlorobenzoamido)-5-{4-[2-oxo-2-(pyrrolidone-1-yl)ethyl]piperazin-1-yl}benzoic acid at a concentration of 6.8 μM-68 μM. Aspergillus flavus )produce.
2. The method according to claim 1, characterized in that, The concentration of the 2-(3-chlorobenzoamido)-5-{4-[2-oxo-2-(pyrrolidone-1-yl)ethyl]piperazin-1-yl}benzoic acid was 68 μM.
3. The method according to claim 1, characterized in that, The aflatoxin is either AFB1 or AFB2. The use of 4,2-(3-chlorobenzoamido)-5-{4-[2-oxo-2-(pyrrolidone-1-yl)ethyl]piperazin-1-yl}benzoic acid in the inhibition of aflatoxin production for non-disease diagnostic and therapeutic purposes, wherein the aflatoxin is produced by Aspergillus flavus ( Aspergillus flavus )produce.
5. The application according to claim 4, characterized in that, The concentration of 2-(3-chlorobenzoamido)-5-{4-[2-oxo-2-(pyrrolidone-1-yl)ethyl]piperazin-1-yl}benzoic acid is 68 μM, and the aflatoxin is AFB1 or AFB2.