CN113712967A

CN113712967A - Application of compound in preparation of anti-aflatoxin B1 toxicity medicine

Info

Publication number: CN113712967A
Application number: CN202111091259.8A
Authority: CN
Inventors: 王旭; 胡嗣祎; 赵书红; 谢胜松; 黄玲利; 彭大鹏; 郝海红; 程古月; 李新云; 张金福
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2021-11-30
Anticipated expiration: 2041-09-17
Also published as: CN113712967B

Abstract

The invention discloses application of a compound with a structural formula shown as the following formula (I) in preparation of a drug for resisting the toxicity of aflatoxin B1. The compound does not influence the growth of cells, but can reduce the toxicity of the aflatoxin B1 to the cells, improve the survival rate of the cells, and reduce the promotion of the oxygen free radical level of the cells and the malondialdehyde level of a lipid peroxidation product caused by the aflatoxin B1, thereby resisting the cytotoxicity of the aflatoxin B1.

Description

Application of compound in preparation of anti-aflatoxin B1 toxicity medicine

Technical Field

The invention belongs to the field of pharmacy, and relates to application of a compound in preparation of a drug for resisting aflatoxin B1 toxicity.

Background

Aflatoxins the mycotoxins produced by some strains of aspergillus flavus and aspergillus parasiticus are a widely-existing mycotoxin and have various derivatives, of which Aflatoxin B1(Aflatoxin B1, AFB1) is the most toxic and carcinogenic. Aflatoxins are mainly found in grain and oil products such as soybeans, corn, peanuts and the like, and are also found in nuts and Chinese herbal medicines. Aflatoxin is polluted in main food crops in China, the pollution to peanut, corn, soybean and grain oil crops is serious, the pollution is serious in animal feed, plant raw materials used by the animal feed are easy to mildew due to improper storage and accumulation operation, and the aflatoxin is easy to generate. AFB1 has strong toxic effect, genetic toxicity and capability of causing hepatocellular carcinoma, and AFB1 has synergistic effect with Hepatitis B Virus (HBV), so that risk of liver cancer can be increased and development of liver cancer can be accelerated. In addition, AFB1 can also promote the development of liver cancer from liver cirrhosis diseases caused by HCV and alcohol. Besides the genotoxicity and carcinogenesis, AFB1 may cause malnutrition, growth retardation and decreased immune function, which seriously affect the health of human beings.

The current prevention and control of aflatoxin B1 mainly depends on controlling the source of aflatoxin, such as preventing AFB1 from generating by improving planting technology and storage method, and degrading AFB1 by physical, chemical and biological methods. In addition, the treatment after the aflatoxin is ingested is also advanced to a certain extent, for example, some known medicines or compounds can resist the toxic action of AFB1, but no specific treatment medicine exists, so that the research and development of novel treatment medicines aiming at the toxic action of AFB1 are very important for the breeding industry and the health of people and livestock.

The applicant finds that a known artificially synthesized small molecular compound has the effect of resisting the toxicity of aflatoxin B1 by using Computer Aided Drug Design (CADD) and molecular simulation docking screening, and no related research report exists at present.

Disclosure of Invention

The invention aims to provide application of a compound in preparing a medicine for resisting the toxicity of aflatoxin B1, and aims to provide a potential medicine for treating AFB1 poisoning for clinic.

The compound of the invention has a structural formula shown as the following formula (I):

in the formula (I), R1 represents an oxygen atom or an alkoxy group; r2 represents H, alkyl, phenylalkyl, benzene, alkoxy-substituted benzene.

Further, the compounds have structural formulas shown in formulas (II) to (V):

most preferably, the compound has a structural formula shown in formula (II), and is named as 1- (1, 3-benzodioxol-5-acyloxy) -3- [4- (2-methoxyphenyl) -1-piperazinyl ] -2-propanol; the English name (1- (1, 3-benzodioxol-5-yloxy) -3- [4- (2-methoxyphenyl) -1-piperazinyl ] -2-propanol); CAS number: 380192-64-3.

The invention verifies the functions of the compounds on various cells, and the results show that:

the compound has no obvious toxic effect on normal cells (porcine kidney cells) and cannot influence the growth of the cells, but can reduce the toxicity of the aflatoxin B1 on the normal cells, improve the survival rate of the cells, and reduce the promotion of the oxygen free radical level of the cells and the malondialdehyde level of lipid peroxidation products caused by the aflatoxin B1, thereby resisting the cytotoxicity of the aflatoxin B1.

Drawings

FIG. 1: effect of compound M2 on cell viability after treatment of PK-15 cells.

FIG. 2: the ability of compound M2 to resist AFB1 cytotoxicity.

FIG. 3: compound M2 lethal PK-15 cell IC to AFB1₅₀The influence of the value.

FIG. 4: effect of compound M2 on the increase in PK-15 cellular oxygen free radical ROS levels caused by AFB 1.

FIG. 5: effect of compound M2 on the rise in the level of Malondialdehyde (MDA), a PK-15 cell lipid peroxidation product, caused by AFB 1. In the figure, P < 0.05.

FIG. 6: the 8 compounds resist the toxic effect of AFB1 on the human hepatoma cell line Huh 7. In the figure, P < 0.001; p < 0.01.

FIG. 7: ability of sesamol to resist AFB1 cytotoxicity. In the figure, P < 0.01.

Detailed Description

The present invention will be described in detail below with reference to specific examples.

Test materials:

1. compound (I)

A total of 8 compounds were used in the experiments, which the applicant designated as M1-8, and which are all known compounds and either synthetically or commercially available. This experiment was purchased from SPECS corporation.

Sesamol was purchased from alatin.

2. Cells

Porcine Kidney cell lines (Pig Kidney 15, PK-15) were purchased from the American Type Culture Collection (ATCC) and stored for use in this laboratory.

The human liver cancer cell line (Huh-7) is from the China type culture Collection of Wuhan university.

3. Reagent

Aflatoxin B1: purchased from Sigama.

DMEM medium: gibico.

Fetal bovine serum: a german PAN.

CCK-8 kit: shanghai Biyuntian Biotechnology Co., Ltd.

ROS kit: jiangsu Kaiyi Biotechnology, Inc.

Example 1: toxic effects of M2 on PK-15 cells

1. Culture of wild-type PK-15 cells

After the logarithmic growth phase of PK-15 cells were digested from a cell culture flask of T25, they were diluted to 10% in a DMEM complete medium containing 10% fetal bovine serum according to cell density⁶Uniformly mixing cells in a cell suspension per mL, inoculating the cells into a 96-well plate by using a discharging gun, adding 100 mu L of the cell suspension into each well, adding PBS (phosphate buffer solution) with the same volume into peripheral wells, slightly beating the periphery of the culture plate to uniformly distribute the cells, and putting the cells into a cell culture box with the temperature of 37 ℃ and the concentration of 5% CO2 after the cells are precipitated at the bottom of the culture plate; and (3) after the cells grow to 50% -60%, absorbing the culture medium, washing the cells once by using sterile PBS, adding a serum-free DMEM culture medium, and absorbing the culture medium after the cell cycle is synchronized after 12 hours.

2. Cell administration

M2 was dissolved in DMSO, then further diluted with DMEM complete medium to a concentration of 2, 4, 8, 16, 20, 24, 28, 32. mu.g/mL, 100. mu.L of DMEM complete medium was added to each well of a 96-well plate, 6 parallel wells were set for each dose group, a blank group (medium alone without cells) and a control group (DMSO was added in the same volume as that of the 20. mu.g/mL drug group) were set simultaneously, PBS was added to the peripheral wells in the same volume, and the wells were placed in an incubator for incubation; and taking out the mixture 36h after adding the medicine for cytotoxicity detection.

CCK-8 detection of M2 cytotoxicity

CCK-8 reagent was mixed with medium at a ratio of 1: the 10 proportions were mixed and added to each well. Put at 37 ℃ with 5% CO₂Culturing for 1h in a cell culture box; the microplate reader reads the absorbance at a wavelength of 450nm (OD 450). The cell viability of the test group relative to the control group was calculated. Cell survival rate ═ cell survival (experimental OD 450-blank OD450)(control OD 450-blank OD 450).

As shown in FIG. 1, M2 had a small effect on PK-15 cells at 2-32. mu.g/mL, and no significant cytotoxicity was observed.

Example 2: detection of M2 ability to resist AFB1 toxicity

Survival curves after co-treatment of cells with AFB1 toxin and M2

(1) PK-15 cells were seeded into 96-well plates at about 10 per well⁵And culturing the cells for 24h, adding medicine after the cells grow to 50-60%, setting the concentration of AFB1 toxin to be 2 mug/mL in each group, setting the concentration of M2 to be 9 concentration gradients between 0.5-40 mug/mL, and diluting the cells in a DMEM culture medium containing 10% fetal calf serum.

(2) The cells are added with drugs, a blank group, a DMSO control, an AFB1 toxin control and an M2 treatment group (9 concentrations) are arranged, each group comprises 6 parallel wells, and after incubation for 36 hours, CCK-8 is used for detecting the survival condition of the cells, wherein the detection method is the same as that of example 1.

Survival curves were obtained for cells co-treated with various concentrations of M2 and AFB1 toxin, as shown in fig. 2, and the survival rate increased with increasing M2 concentration, indicating that M2 has a lethal effect against AFB 1.

M2 vs AFB1 toxin IC₅₀Influence of the value

(1) PK-15 cells were seeded into 96-well plates at about 10 per well⁵And culturing the cells for 24h, adding medicine after the cells grow to 50-60%, and setting the concentration gradient of AFB1 toxin to be 0, 1, 2, 4, 6, 8, 12, 16 and 20 mu g/mL. According to the results of FIG. 2, the optimal concentration of M2 was 24. mu.g/mL, and the optimal concentration was diluted in 10% fetal bovine serum in DMEM medium.

(2) And (3) adding medicine to treat cells, setting an AFB1 group and an M2+ AFB1 group, setting 6 parallel holes for each concentration, and detecting the survival condition of the cells by CCK-8 after incubation for 36h, wherein the detection method is the same as that of example 1, and an IC5 curve of AFB1 and an IC50 curve of AFB1 under the action of M2 are determined.

The results are shown in FIG. 3, and the half lethal concentration of AFB1 toxin to PK-15 cells is 1.215 μ g/mL when M2 is not added; the half-lethal concentration of AFB1 toxin on PK-15 cells increased to 4.149 μ g/mL when M2 was added, further demonstrating the effect of M2 against AFB1 toxicity.

Example 3: m2 reduction of AFB1 results in an increase in PK-15 cellular oxygen free Radical (ROS) levels

AFB1 toxin with M2 treated cells.

(1) PK-15 cells are inoculated into a 12-well plate and cultured for 24h, and after the cells grow to 40-50%, the cells are treated by adding medicines. AFB1 toxin concentration was fixed at 0.1. mu.g/mL, M2 concentration was set at 2. mu.g/mL, diluted in 10% fetal bovine serum in DMEM medium.

(2) Adding medicine to treat cells, arranging a zeroing group, a blank group, an AFB1 group, an M2 group and an AFB1+ M2 group, wherein each group comprises 2 parallel holes, and carrying out fluorescent staining treatment after 48 hours of treatment.

ROS detection reagent treatment

(1) The fluorescent probe DCFH-DA is used for detecting active oxygen, and the active oxygen in the cells can oxidize the non-fluorescent DCFH to generate fluorescent DCF and emit green fluorescence.

(2) DCFH-DA was diluted in serum-free medium at a final concentration of 10. mu.M at a ratio of 1: 1000.

(3) Discarding the culture medium, washing for 2 times by PBS, digesting the cells by pancreatin, adding a basic culture medium, fully blowing to prepare a cell suspension, centrifuging to obtain cell precipitates, collecting the cells, suspending the cells in diluted DCFH-DA, incubating for 20 minutes in a cell culture box at 37 ℃, wherein the cell concentration is one million to two million/ml. Mix by inversion every 3-5 minutes to bring the probe and cells into intimate contact. Cells were washed three times with serum-free cell culture medium to remove DCFH-DA well without entering the cells.

3. Flow cytometry ROS (reactive oxygen species) positive rate detection method

Detection is carried out by using an analytical flow cytometer, green fluorescence is detected under an FITC channel, and the proportion of cells with the green fluorescence represents the ROS positive rate. The fluorescence intensity of the FITC peak value of the cells of the control group is used as a median as a standard line, the FITC-A + on the right side represents ROS positive, the lower value represents the proportion of the cells with fluorescence intensity higher than the median, and the increase of the value represents the increase of ROS positive rate, so that the increase of the ROS level of the cells is indicated.

The results are shown in FIG. 4. The FITC-A + value of the cells in the AFB1 group is increased from 51 to 78.1, which shows that AFB1 causes the ROS level of the cells to be increased, the oxidative stress level of the cells is increased, while the cells in the M2 group are only increased from 42.7 to 55.5 under the action of AFB1, and the positive rate of the cells is similar to that of the blank group, which shows that M2 can effectively prevent the ROS level of the cells from being increased due to AFB 1.AFB1 caused an increase in PK-15 cell Reactive Oxygen Species (ROS) levels, while M2 alleviated the ROS level increase caused by AFB 1.

Example 4: m2 lowering AFB1 resulted in elevated levels of the PK-15 cell lipid peroxidation product Malondialdehyde (MDA)

AFB1 toxin and M2 treated cells

(1) And (3) inoculating the PK-15 cells into a 6-well plate, culturing for 24h, and adding medicine after the cells grow to 60-70%. AFB1 toxin concentration was fixed at 2. mu.g/mL, M2 concentration was set at 10. mu.g/mL, diluted in DMEM medium with 10% fetal bovine serum.

(2) Adding medicine to treat cells, setting blank group, AFB1 group, M2 group and AFB1+ M2 group, and extracting cell protein after treating for 36 h.

2. Extraction of cellular proteins

(1) The cells were removed from the incubator, the medium was discarded, and the cells were washed twice with PBS and discarded.

(2) Preparing cell lysate, adding a phosphatase inhibitor, a protease inhibitor and an EDTA solution into the cell lysate, and preparing into a cell protein extracting solution.

(3) Adding 100 μ L cell protein extract into each well of cell, standing on ice for 10min, blowing off the cell, transferring into 1.5mL EP tube, lysing on ice for 40min, centrifuging at 4 deg.C 12000g/min for 10min to precipitate cell debris, and collecting supernatant as cell whole protein.

(4) Protein concentration was determined using the BCA kit.

2. Detection of cellular MDA levels

The cellular MDA level was measured using a micro Malondialdehyde (MDA) assay kit purchased from Jiangsu Kai Bio-technology GmbH under the cat number KGT 004-1.

Comparing the groups of AFB1, M2 and AFB1+ M2 with the blank group of MDA as a reference, M2 was found to down-regulate the increase of cellular MDA level caused by AFB1, as shown in fig. 5.

Example 5: expression of the compound against AFB1 on other cells (human hepatoma cell line)

Huh7 cells were plated in 6-well plates, approximately 10 cells per well⁵And culturing the cells for 24h, and adding medicine after the cells grow to 50-60%, wherein the concentration of AFB1 toxin is set to be 4 mu g/mL in each group, and the concentration of 8 compounds such as M1-8 is 10 mu g/mL.

Adding medicine to treat cells, setting a blank group, an AFB1 toxin control group and a compound treatment group, setting 6 parallel holes in each group, and detecting the survival condition of the cells by CCK-8 after incubation for 36 h.

The obtained 8 compounds have the capacity of resisting AFB1 lethal Huh7 cell effects, and as shown in figure 6, the four compounds of M2, M4, M5 and M6 have strong resistance to the cytotoxicity of AFB1 toxin. The four compounds all have 1, 3-benzodioxole, piperazine and propanol groups, which show that the three groups are functional groups of the compounds and play an important role in the functions of the compounds.

Example 6: sesamin expression on PK-15 cells against AFB1

Sesamol (3, 4-methylenedioxyphenol) has been reported to have antioxidant capacity, and we speculate that sesamol may have the capacity of resisting AFB1 cytotoxicity, so we also tested the function of sesamol, and the test method is as follows:

PK-15 cells were seeded into 96-well plates at about 10 per well⁵And culturing the cells for 24 hours, adding medicine after the cells grow to 50-60%, setting the concentration of AFB1 toxin to be 2 mug/mL in each group, setting the concentration of sesamol to be 6 concentration gradients between 32-1024 mug/mL, and diluting the cells in a DMEM culture medium containing 10% fetal calf serum.

Adding medicine to treat cells, setting a blank group, an AFB1 toxin control group, an AFB1 toxin and sesamol (6 concentrations) treatment group, setting 6 parallel holes in each group, and detecting the survival condition of the cells after incubation for 36 h.

As shown in FIG. 7, the survival rate of PK-15 cells reached 80% when the sesamol concentration was 512. mu.g/mL, whereas the survival rate of PK-15 cells reached 100% when the compound of the present invention was 20-30. mu.g/mL, indicating that the activity of the compound of the present invention was much higher than that of sesamol.

Claims

1. The application of the compound with the structural formula shown as the following formula (I) in preparing the anti-aflatoxin B1 toxic medicine,

2. The use of claim 1, wherein the compound has the structural formula shown in formulas (ii) - (v):

3. use according to claim 2, characterized in that: the structural formula of the compound is shown as a formula (II), and the name of the compound is 1- (1, 3-benzodioxol-5-acyloxy) -3- [4- (2-methoxyphenyl) -1-piperazinyl ] -2-propanol; the English name (1- (1, 3-benzodioxol-5-yloxy) -3- [4- (2-methoxyphenyl) -1-piperazinyl ] -2-propanol); CAS number: 380192-64-3.

4. Use according to any one of claims 1 to 3, wherein: the compound does not influence the growth of cells, but can reduce the toxicity of the aflatoxin B1 to the cells, improve the survival rate of the cells, and reduce the promotion of the oxygen free radical level of the cells and the malondialdehyde level of a lipid peroxidation product caused by the aflatoxin B1, thereby resisting the cytotoxicity of the aflatoxin B1.