Application of disulfide bond compounds in the prevention and control of microbial diseases

Abstract

The invention belongs to the field of medicinal chemistry and discloses the use of disulfide bond compounds in preventing and treating diseases caused by rapeseed sclerotinia, solani, tomato gray mold, wheat fusarium, cotton wilt, rice blast, Aspergillus flavus, Aspergillus niger, citrus canker, potato blackleg, Escherichia coli and Candida albicans. Some of the compounds show higher inhibitory effects and can be used to develop bactericidal lead compounds.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, discloses a new use of a disulfide bond compound, and specifically relates to a use of the disulfide bond compound in preventing and treating diseases caused by rapeseed sclerotinia, solani, tomato gray mold, wheat fusarium, cotton wilt, rice blast, Aspergillus flavus, Aspergillus niger, citrus canker, potato black shank, Escherichia coli and Candida albicans. Background Art

[0002] Over the past seven decades, China's rice production has gradually shifted northward as new arable land has emerged due to rising temperatures. Also due to climate change, corn farmers in the Midwest of the United States are adopting new farming practices, including crop diversification, agricultural intensification, and even abandoning corn cultivation. Pathogens and pests are the main causes of crop losses, with an estimated average loss of 17.2% to 30.0% of major crops worldwide. In addition, about a quarter of the world's grain harvest is lost each year due to fungal diseases. Aspergillus niger can cause plant diseases, causing plants to mold, further causing agricultural losses, not only onions, but also peanuts and grapes. Post-harvest disasters. Chinese medicinal materials and decoction pieces are also susceptible to fungal contamination during production, processing, storage and transportation, producing hepatotoxic toxin residues such as aflatoxin and ochratoxin A, which seriously affects the quality and medication safety of Chinese medicinal materials, decoction pieces and Chinese medicine preparations. Due to the influence of storage conditions, such as high temperature, high humidity, and lack of ventilation, peanuts are easily contaminated by Aspergillus flavus and produce aflatoxin, causing huge economic losses. Aflatoxin is a type of fungal toxin that can cause cancer, teratogenesis, and mutagenesis. If people eat peanuts and their products containing aflatoxin, it will endanger human health. Mold damage not only occurs in foods in the refrigerator, but is also ubiquitous from crop growth to storage and processing, and is a major problem in grain production and food processing. Therefore, the prevention and control of mold diseases is also particularly important. In order to effectively control plant diseases, people have adopted various measures, including crop rotation, seed detoxification, cross-protection of weak strains between viruses, selection of disease-resistant varieties, control of virus vectors, and use of chemical pesticides. In recent years, there have been new advances in the study of disease resistance of transgenic plants. However, these measures cannot effectively overcome the harm of pathogens. Modern plant protection is mainly based on chemical control, which is efficient, cheap and convenient. However, excessive use of chemical pesticides will cause pests to develop resistance and cause serious pesticide residue problems. Under the current premise of vigorously advocating green food and environmental protection, strengthening the comprehensive prevention and control of plant diseases and reducing the use of chemical pesticides have become one of the key contents of the work of plant protection workers. In addition, Escherichia coli disease is an infectious disease caused by the pathogen Escherichia coli. It mainly relies on the bacteria and its metabolites to cause diseases, such as diarrhea, sepsis, etc. It has caused huge economic losses in human health and poultry farming such as chickens, ducks, and pigs. Candida albicans (commonly known as white Candida) is an important conditional pathogenic fungus, which usually exists in the normal human oral cavity, upper respiratory tract, intestines and vagina. Generally, there are few in the normal body and it does not cause disease, but when the body's immune function or general defense power decreases or the normal flora structure is out of balance, it will turn into a pathogenic bacteria and cause harm to the human body. According to statistics, there are 75 million cases of female vaginal infection caused by Candida, 13 million cases of thrush, and more than 400,000 people with blood and deep organ infections each year.Therefore, in order to develop fungicides that can effectively control diseases, researchers are constantly looking for and screening new bio-based antimicrobial substances, especially plant-based fungicides, to avoid the risks to the environment and health caused by chemical drugs at high concentrations, while not affecting the effectiveness of disease prevention and control. Therefore, the development of new, fast and effective active molecules is crucial to preventing the spread of pathogens and crop diseases, ecological planting and organic food, and reducing economic losses in agricultural planting and poultry farming.

[0003] Disulfide bonds (SS bonds) are an important chemical functional group, which mainly exist in various natural small molecule compounds and biological protein structures. Some small molecule compounds containing disulfide bonds show rich biological activities, such as the familiar lipoic acid with antioxidant activity and nereis toxin with acetylcholine receptor inhibitory activity. In the body, the main functions of disulfide bonds include helping protein structure solidification, controlling cell signal transduction, and participating in metabolic pathways. Since disulfide bonds have low toxicity in the body and can break in the presence of reduced glutathione (GSH) in the external environment, many drug developers introduce disulfide bonds into drug structures to achieve better therapeutic effects. Organic sulfur compounds in onion vegetables such as garlic and onions can inhibit Helicobacter pylori in the gastrointestinal tract, thereby reducing the incidence of gastric cancer. In addition, increasing the intake of 20 grams of onions and garlic every day can reduce the incidence of gastric cancer by 10%. Based on this, the present invention takes disulfide bond compounds as the research object and evaluates their fungicidal activity. It is found that disulfide bond compounds show certain inhibitory effects on rapeseed Sclerotinia sclerotiorum, Rhizoctonia solani, tomato Botrytis cinerea, wheat fusarium wilt, cotton wilt, rice blast, Aspergillus flavus, Aspergillus niger, citrus canker, potato black shank, Escherichia coli, and Candida albicans. In particular, compound 11 has a strong antibacterial activity and can be further developed as a fungicidal lead compound. Summary of the invention

[0004] The purpose of the present invention is to address the defects in the prior art, provide a natural source fungicide for agricultural production, food preservation and human health, and use the disulfide bond compound in preventing and controlling microbial diseases.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical method: a disulfide bond compound for preventing and controlling diseases caused by rapeseed sclerotinia, Rhizoctonia solani, tomato gray mold, wheat fusarium, cotton wilt, rice blast, Aspergillus flavus, Aspergillus niger, citrus canker, potato black shank pathogen, Escherichia coli, and Candida albicans, namely compounds 1 to 18, whose structural formulas are shown in 1-18.

[0006] As a new type of fungicide, disulfide bond compounds have the following advantages:

[0007] 1) High fungicidal activity. In particular, compound 11 has an EC 50 At 6.117-25.091 μg / mL, it can be developed as a new bactericidal lead.

[0008] 2) Broad fungicide spectrum. In particular, compound 11 has strong fungicidal activity against rapeseed sclerotinia, Rhizoctonia solani, tomato gray mold, wheat fusarium, cotton wilt, rice blast, Aspergillus flavus, Aspergillus niger, citrus canker, potato black shank, Escherichia coli, and Candida albicans.

[0009] 3) Some disulfide bond compounds are natural active ingredients. As fungicides, they are new, highly effective, low-toxic, environmentally friendly, and not prone to resistance. They can be developed into new pesticides for the production of green, pollution-free, and sustainable agricultural products, and are new biological antimicrobial agents. DETAILED DESCRIPTION

[0010] In order to better understand the present invention, the above contents of the present invention are further described in detail below through specific implementation modes, but this should not be construed as limiting the present invention.

[0011] Example 1: Compounds 1-18 were isolated and their structures were identified in accordance with those reported in the literature.

[0012] 2,2'-disulfide dipyridine (1) CAS: 2127-03-9, purity > 98%; dibenzothiazole disulfide (2) CAS: 120-78-5, purity > 98%; dibenzyl disulfide (3) CAS: 150-60-7, purity > 98%; 3,3',5,5'-tetrachlorodiphenyl disulfide (4) CAS: 137897-99-5, purity > 99%; dipropyl disulfide (5) CAS: 629-19-6, purity>99%; a-lipoic acid (6) CAS: 1077-28-7, purity>99%; diphenyl disulfide (7) CAS: 882-33-7, purity>99%; 2,2'-difluorodiphenyl disulfide (8) CAS: 14135-38-7, purity>98%; 4,4'-dimethoxydiphenyl disulfide (9) CAS: 5335-87-5, purity>98%; 4, 4'-Bipyridyl disulfide (10) CAS: 2645-22-9, purity > 98%; 3,3'-dihydroxydiphenyl disulfide (11) CAS: 21101-56-4, purity > 98%; bis(2-thienyl) disulfide (12) CAS: 6911-51-9, purity > 95%; isopropyl disulfide (13) CAS: 4253-89-8, purity > 98%; difurfuryl disulfide (14) C AS: 4437-20-1, purity>95%; diethyl disulfide (15) CAS: 110-81-6, purity>99%; 3,3'-difluorodiphenyl disulfide (16) CAS: 63930-17-6, purity>95%; diisoamyl disulfide (17) CAS: 2051-04-9, purity>98%; dibutyl disulfide (18) CAS: 629-45-8, purity>95%.

[0013] Example 2: Indoor antifungal activity determination and results

[0014] The plant pathogenic fungi used in this experiment were laboratory preserved strains, and the culture media used were potato agar glucose medium (PDA for short) and YPG medium.

[0015] PDA medium formula: 200 g potato (peeled), 20 g glucose, 15 g agar, 1000 mL distilled water, natural pH.

[0016] Preparation method of PDA culture medium: Wash and peel the potatoes, weigh 200g and cut into small pieces, boil in distilled water for about 20min (the potato pieces are soft but not mushy), filter with eight layers of gauze, make up the filtrate to 1000mL with distilled water, add 15g agar and 20g glucose, stir to fully dissolve, divide into Erlenmeyer flasks, sterilize at 121℃ for 20min, cool and set aside.

[0017] YPG medium formula: agar 20g, glucose 5g, peptone 5g, yeast extract 5g, distilled water 1000mL, natural pH.

[0018] Preparation method of YPG medium: add the ingredients into the beaker in sequence, stir to fully dissolve them, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and set aside after cooling.

[0019] Experimental methods

[0020] 1) Mycelial growth rate method was used.

[0021] 1. Activation of bacterial strains: Culture the plant pathogens on PDA plates at 25°C for 3 to 6 days.

[0022] 2. Preparation of drug plates: Heat and melt the PDA culture medium, cool it to 45-50°C, and add different concentrations of disulfide bond compounds to prepare drug-containing plates.

[0023] 3. Inoculation culture: In a clean bench, use a hole puncher to punch a bacterial cake (5 mm in diameter) at the edge of the hyphae cultured for 3 to 6 days (the growth conditions should be as consistent as possible), then use an inoculation needle to pick it to the center of the medicine plate, and then invert it in an incubator (25°C) for culture.

[0024] 4. Result determination: After the mycelium of the blank control group was fully grown, the growth diameter of the mycelium of the drug-treated group was measured by the cross method, and the inhibition rate was calculated.

[0025] 5. Inhibition rate (%) = (control mycelium diameter - treated mycelium diameter) / (control mycelium diameter - cake diameter) × 100

[0026] 6. Set up 3 parallel experiments for each concentration.

[0027] Table 1 Antibacterial activity of disulfide compounds against plant pathogenic fungi

[0028]

[0029] Note: Each treatment was repeated three times in the experiment, and the data in the table are the average values ​​of the three repetitions.

[0030] From the test results in Table 1, it can be seen that the disulfide bond compound of the present invention exhibits good inhibitory activity against rapeseed sclerotinia, Rhizoctonia solani, tomato gray mold, wheat fusarium, cotton wilt, rice blast, Aspergillus flavus, and Aspergillus niger. In summary, the disulfide bond compound of the present invention has a simple structure and exhibits good inhibitory activity against agricultural pathogens and pathogens in food preservation, and is worthy of further research.

[0031] Example 3: Indoor antibacterial activity determination and results of plant pathogenic bacteria and human pathogenic bacteria

[0032] The plant pathogenic bacteria used in this experiment were kept at -80℃ in the laboratory, and the MIC values ​​were determined by the two-fold dilution method. The plant pathogenic bacteria used in the laboratory were all cultured in nutrient broth (MH for short), and human pathogens were cultured in YEPD medium.

[0033] MH medium formula: MH broth powder 21g, distilled water 1000mL, natural pH.

[0034] Preparation method of MH medium: weigh 21g MH broth powder into 1000mL distilled water, stir to fully dissolve it, divide it into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and set aside after cooling.

[0035] YEPD medium formula: yeast extract 10g, peptone 20g, glucose 20g, 2mg / mL chloramphenicol aqueous solution, natural pH.

[0036] YEPD medium preparation formula: weigh 10g yeast extract, 20g peptone, 20g glucose, add 900ml triple distilled water and stir to fully dissolve, add 50ml 2mg / mL chloramphenicol aqueous solution, dilute to 1000ml. Dispense in Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, cool and store at 4℃ for later use.

[0037] Experimental methods

[0038] 1) Use the two-fold dilution method.

[0039] 1. Add 50 μL of MH broth medium to each well of a 96-well plate.

[0040] 2. Add 150μL of the prepared drug solution to the first well of the 1 / 2 / 3 columns, and then dilute the drug twice. That is, after adding the drug solution to the first well, use a pipette to blow it thoroughly (at least 3 times) to mix the drug and broth thoroughly, then pipette 100μL and add it to the second well and blow it thoroughly. Repeat this until the last well, and pipette 100μL from the 8th column and discard it.

[0041] 3. Add 50 μL of diluted bacterial solution to each well and repeat 3 times.

[0042] 4. On the same plate, columns 1 / 2 / 3 are the treatment groups, and columns 4 / 5 are the negative controls (only blank broth is added without bacterial solution).

[0043] 5. Place the 96-well plate in a 37°C constant temperature incubator for 16 to 20 hours, observe the results, and measure the absorbance of each well with an enzyme-labeled instrument.

[0044] 6. Inhibition rate (%) = [absorbance value of negative control - absorbance value of blank control - (absorbance value of drug administration group - absorbance value of reference control)] / (absorbance value of negative control - absorbance value of blank control) × 100

[0045] 7. The results of plant pathogenic bacteria and human pathogenic bacteria are shown in Table 2.

[0046] Table 2 Antibacterial activity of disulfide compounds against plant pathogenic bacteria and human pathogenic bacteria

[0047]

[0048] From the test results in Table 2, it can be seen that the disulfide bond compounds of the present invention exhibit certain inhibitory activity against citrus canker solanum, potato black leg fungus, Escherichia coli and Candida albicans, especially compounds 1, 10, 11 and 12 exhibit excellent antibacterial activity.

Claims

1. Use of a disulfide bond compound in preventing and controlling microbial diseases, characterized in that: The disulfide bond compound has the following molecular structure: The microbial diseases are any one of the diseases caused by Sclerotinia sclerotiorum of rape, Botrytis cinerea of ​​tomato, Gibberellic acid of wheat, Fusarium wilt of cotton, Rice blast, citrus canker and potato blackleg pathogen.

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