Application of main secondary metabolites of traditional Chinese medicine fructus psoraleae in prevention and treatment of agricultural pathogenic bacteria

Abstract

The invention discloses application of main secondary metabolites of traditional Chinese medicine fructus psoraleae in prevention and treatment of agricultural pathogenic bacteria, and relates to the technical field of natural medicinal chemistry. Main secondary metabolites, namely psoralen, bakuchiol, psoralen A and psoralen B, of the traditional Chinese medicine fructus psoraleae have excellent antibacterial activity on rice bacterial blight pathogenic bacteria ACCC 11602, citrus canker pathogenic bacteria, ralstonia solanacearum, pseudomonas solanacearum, xanthomonas campestris and pseudomonas syringae; the compound is expected to be developed into a novel antibacterial agent.

Description

Technical Field

[0001] The invention relates to the technical field of natural drug chemistry, and in particular to application of main secondary metabolites of traditional Chinese medicine psoralea corylifolia in preventing and treating agricultural pathogenic bacteria. Background Art

[0002] Plant bacterial diseases are the most difficult to control during the growth of crops, causing serious damage to crops and huge economic losses. For example, rice bacterial blight can cause a 20% to 50% reduction in rice production, and citrus canker seriously threatens important citrus fruit crops worldwide. Since the 1960s, the large-scale use of chemical pesticides has been the main method for pest control. Although pesticides have been successful in reducing pest populations, their application has had a negative impact on the environment and the crops themselves. Therefore, research and development of new high-efficiency, low-toxic, and low-residue pesticides that can effectively protect and treat plants is one of the main goals of pesticide development.

[0003] Natural products are a class of substances with specific active substances, most of which have good antibacterial activity. Natural products have always been an important source of potential lead drugs. In the past two decades, more than 1 / 3 of the drugs approved by the FDA for marketing are natural products and their derivatives. Compared with traditional chemically synthesized molecules, natural products have rich skeleton diversity and structural complexity, showing great advantages in new drug discovery. Therefore, natural products are a treasure trove of drug development worthy of attention and have great potential for drug acquisition.

[0004] Psoralea corylifolia L. is a dried mature fruit of the leguminous plant Psoralea corylifolia L. Psoralen, psoralen, bakuchiol, psoralen A, and psoralen B are effective ingredients extracted from the plant Psoralea corylifolia, which have anti-cancer, anti-inflammatory, antibacterial, antidepressant and other effects. Among them, bakuchiol is the main component of the volatile oil of the traditional Chinese medicine Psoralea corylifolia, which is a prenylphenol terpene compound. Psoralen and psoralen are coumarin compounds extracted from the traditional Chinese medicine Psoralea corylifolia, and psoralen A and psoralen B are flavonoid compounds extracted from the traditional Chinese medicine Psoralea corylifolia. The present invention uses psoralen, bakuchiol, psoralen A, and psoralen B extracted and separated from Psoralea corylifolia to evaluate their antibacterial activity against agricultural pathogenic bacteria. Therefore, the present invention aims to illustrate the new antibacterial uses of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia and provide new antibacterial strategies to fill the gap in the types of fungicides on the market and cope with the current agricultural crisis. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides the use of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia in preventing and treating agricultural pathogenic bacteria, which solves the problem of the current lack of efficient antibacterial agents. The specific technical scheme is as follows:

[0006] The invention provides use of a main secondary metabolite of a traditional Chinese medicine, psoralea corylifolia, in preventing and treating agricultural pathogenic bacteria.

[0007] Furthermore, the main secondary metabolites of the Chinese medicine psoralea corylifolia include: psoralen, bakuchiol, psoralen A and psoralen B;

[0008] The molecular structural formula of the psoralen is as follows:

[0009]

[0010] The molecular structural formula of the bakuchiol is as follows:

[0011]

[0012] The molecular structural formula of the psoralen is as follows:

[0013]

[0014] The molecular structural formula of the psoralen is as follows:

[0015]

[0016] Furthermore, the agricultural pathogenic bacteria include: Xanthomonas oryzaeACCC 11602, the pathogen of rice bacterial blight, Xanthomonas axonopodis, the pathogen of citrus canker, Ralstonia solanacearum, Pseudomonas solanacearum, Xanthomonas campestris pv. Campestris, and Pseudomonas syringae.

[0017] The invention also provides a medicament for preventing and treating plant bacterial diseases, wherein the medicament contains the main secondary metabolites of the traditional Chinese medicine psoralea corylifolia.

[0018] Furthermore, the weight percentage of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia in the medicament is 1 to 99%.

[0019] Furthermore, the agent also contains an adjuvant or an agriculturally acceptable carrier.

[0020] Furthermore, the dosage form of the medicament is a suspension, a dispersible oil suspension, a dry suspension, a wettable powder, an emulsifiable concentrate or a water-dispersible granule.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention found that psoralen, bakuchiol, psoralen A and psoralen B all have good antibacterial activity against agricultural pathogenic bacteria. Among them, the MIC value of bakuchiol against citrus canker pathogens and Ralstonia solanacearum is 3.12μg / mL, and the MIC value against rice bacterial blight bacteria ACCC 11602 is 12.5μg / mL; the MIC values ​​of psoralen A against wild campestris are 100μg / mL and 25μg / mL, respectively, which are better than the positive control drug thiophanate-copper; the MIC value of psoralen B against citrus canker pathogens and Ralstonia solanacearum is 1.56μg / mL, the MIC value against rice bacterial blight bacteria ACCC 11602 and Pseudomonas solanacearum is 6.25μg / mL, and the MIC value against wild campestris is 25μg / mL.

[0023] 2. The present invention found that the main secondary metabolites of the tested traditional Chinese medicine Psoralea corylifolia have simple structures and are safe to use, especially bakuchiol and psoralen, which have excellent antibacterial effects and are expected to be developed into new antibacterial agents. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. The experimental methods described in the following examples are conventional methods unless otherwise specified. The main secondary metabolites of the Chinese medicine Psoralea corylifolia and various auxiliary agents are all commercially available.

[0025] Example 1: Indoor antibacterial activity determination of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia against agricultural pathogenic bacteria

[0026] 1) Test drugs: the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia, including psoralen, bakuchiol, psoralen A and psoralen B.

[0027] 2) Test bacteria: Rice bacterial blight ACCC 11602, citrus canker pathogen, Ralstonia solanacearum, Pseudomonas solanacearum, Xanthomonas campestris, and Pseudomonas syringae.

[0028] 3) Antimicrobial activity test:

[0029] The strain used in this experiment is a strain frozen at -80℃ in the laboratory with 30% glycerol. Take out the frozen strain, streak it on NB solid culture medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, agar: 15g, distilled water: 1L, pH7.0; sterilize at 121℃ for 20min), and culture it at a constant temperature of 28℃ until a single colony grows. Pick up the single colonies on the solid culture medium and transfer them to NB liquid culture medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, distilled water: 1L; sterilize at 121℃ for 20min), and culture it on a constant temperature shaker at 28℃ and 180rpm under shaking conditions until it reaches the logarithmic growth phase. Dilute the strain in the logarithmic growth phase to about 10 with NB liquid culture medium. 6 Dissolve the compounds in DMSO, add them to the liquid culture medium, and mix them evenly to prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of the medium containing about 10 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, and the final drug concentration was 100μg / mL. 100μL of bacterial solution of the same concentration containing an equal amount of DMSO was used as a control. The 96-well plate was cultured in a 28℃ constant temperature incubator for 24-48h until the bacterial solution of the control group grew, and the OD of the bacterial solution in the well was measured at 600nm using an ELISA reader. 600 In addition, the OD values ​​of 100 μL of liquid culture medium and the drug with a concentration of 100 μg / mL were measured to correct the OD values ​​caused by the culture medium and the drug itself. The calculation formula for the corrected OD value and inhibition rate is as follows:

[0030] Corrected OD 600 =OD of culture medium containing bacteria 600 - Axenic culture OD 600 ;

[0031]

[0032] The four main secondary metabolites of the Chinese herbal medicine Psoralea corylifolia described in this patent and the positive control drug thiophanate-methyl (purity of 20%, purchased from Zhejiang Longwan Chemical Co., Ltd.) were selected to determine the inhibitory effects on rice bacterial blight pathogen ACCC 11602, citrus canker pathogen, Ralstonia solanacearum, Pseudomonas solanacearum, Xanthomonas campestris and Pseudomonas syringae. All experiments were repeated three times, and the inhibition rates of the compounds determined are shown in Table 1.

[0033] Table 1. Antibacterial effects of the main secondary metabolites of Psoralea corylifolia against agricultural pathogenic bacteria

[0034]

[0035] Note: The initial determination concentration of the positive control drug activity is 400 μg / mL; “-” means that the inhibition rate of the compound on the strain was not measured.

[0036] The compound-containing liquid culture medium was diluted by two-fold dilution in a 96-well plate to obtain 50 μL of a series of concentrations of the drug-containing culture medium and the inhibition rate corresponding to the series of concentrations was determined according to the same experimental method as above. All experiments were set up in triplicate to determine the MIC of the compound. 90 The values ​​(the lowest drug concentration that inhibits 90% bacterial growth) are shown in Table 2.

[0037] Table 2. MIC values ​​(μg / mL) of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia against agricultural pathogenic bacteria

[0038]

[0039] It can be seen from the test results in Tables 1 and 2 that the bakuchiol and psoralen involved in the present invention have excellent antibacterial activity against the tested agricultural pathogenic bacteria. The MIC value of bakuchiol against the pathogenic bacteria of citrus canker and Ralstonia solanacearum is 3.12 μg / mL, and the MIC value against the rice bacterial blight bacteria ACCC 11602 is 12.5 μg / mL. The MIC value of psoralen against the pathogenic bacteria of citrus canker and Ralstonia solanacearum is 1.56 μg / mL, the MIC value against the rice bacterial blight bacteria ACCC 11602 and Pseudomonas solanacearum is 6.25 μg / mL, and the MIC value against Xanthomonas campestris is 25 μg / mL. Psoralen has a certain antibacterial activity against Xanthomonas campestris, with a MIC value of 25 μg / mL, which is better than the positive control drug thiophanate-copper.

[0040] Preparation Example

[0041] Preparation Example 1

[0042] 40% Psoralea corylifolia wettable powder

[0043] Components content(%) Remark Psoralen 40 Active ingredients Sodium dodecylbenzenesulfonate 5 Wetting Agents Sodium methylnaphthalene sulfonate formaldehyde condensate 1.5 First dispersant Sodium lignin sulfonate 3 Second dispersant White Carbon Black 3 filler diatomite Make up 100 filler

[0044] The active ingredient, wetting agent, dispersant and filler are weighed according to the proportion, added into a food processor and mixed evenly, and then put into a jet mill for grinding to obtain the product.

[0045] Preparation Example 2

[0046] 25% Bakuchiol EC

[0047]

[0048]

[0049] The above active ingredients, emulsifiers and solvents are weighed according to the proportions, mixed and stirred evenly to obtain a uniform liquid, which is 25% bakuchiol emulsifiable concentrate.

[0050] Preparation Example 3

[0051] 30% Bakuchiol EC

[0052] Components content(%) Remark Bakuchiol 30 Active ingredients Lauryl alcohol polyoxyethylene ether 4 Emulsifier Twain 60 3 Emulsifier Xylene 20 Solvents

[0053] The above active ingredients, emulsifiers and solvents are weighed according to the proportions, mixed and stirred evenly to obtain a uniform liquid, which is 30% bakuchiol emulsifiable concentrate.

[0054] Preparation Example 4

[0055] 45% Psoralen A WP

[0056] Components content(%) Remark Psoralen 45 Active ingredients Sodium dodecylbenzenesulfonate 5 Wetting Agents Sodium methylnaphthalene sulfonate formaldehyde condensate 1.5 First dispersant Sodium lignin sulfonate 3 Second dispersant White Carbon Black 3 filler Kaolin Make up 100 filler

[0057] The active ingredient, wetting agent, dispersant and filler are weighed according to the proportion, added into a food processor and mixed evenly, and then put into a jet mill for grinding to obtain the product.

[0058] Preparation Example 5

[0059] 20% Psoralen Suspension

[0060]

[0061]

[0062] The laboratory preparation method of the suspension concentrate is as follows: weigh the wetting agent, dispersant, antifreeze, preservative, defoamer, and deionized water according to the ratio, add them into a beaker and mix them evenly, then add the active ingredient into the beaker, and then transfer it to a sand mill, add zirconium oxide beads into the sand mill, grind for 2-3 hours, add thickener xanthan gum and magnesium aluminum silicate and continue grinding for 0.5 hours, control the particle size D95 below 5 microns, filter to remove the zirconium oxide beads, and the suspension concentrate can be obtained.

[0063] Dosage Form Preparation Example 6

[0064] 45% Psoralen water dispersible granules

[0065] Components content(%) Remark Psoralen 60 Active ingredients Alkylaminotaurine 2.5 Wetting Agents Sodium lignin sulfonate 3.2 First dispersant Sodium naphthalenesulfonate 2.6 Second dispersant Sodium sulfate 2 Disintegrants Polyvinylpyrrolidone 2 Binder Kaolin Make up 100 filler

[0066] The laboratory preparation method of water-dispersible granules is as follows: psoralen, wetting agent, dispersant, disintegrant, binder and filler are weighed according to the proportion, added into a food processor and mixed evenly, then put into a jet mill for grinding, mixed again with a food processor, added with an appropriate amount of water, and extruded into granules with a granulator to obtain the granules.

[0067] Preparation Example 7

[0068] 50% Psoralen A Suspension

[0069] Components content(%) Remark Psoralen 50 Active ingredients Alkylbenzene polyoxyethylene ether phosphate 3.5 Wetting Agents Polyarylphenol polyoxyethylene ether 6 First dispersant Sodium polycarboxylate 2 Second dispersant glycerin 2 antifreeze Xanthan gum 2 Thickener benzoic acid 0.5 preservative Silicone 0.5 Defoaming agent Deionized water Make up 100 filler

[0070] The laboratory preparation method of the suspension concentrate is as follows: weigh the wetting agent, dispersant, antifreeze, preservative, defoamer, and deionized water according to the ratio, add them into a beaker and mix them evenly, then add the active ingredient into the beaker, and then transfer it to a sand mill, add zirconium oxide beads into the sand mill, grind for 2-3 hours, add thickener xanthan gum and continue grinding for 0.5 hours, so that the particle size D95 is controlled below 5 microns, filter to remove the zirconium oxide beads, and the suspension concentrate can be obtained.

[0071] Preparation Example 8

[0072] 30% Psoralen B WP

[0073] Components content(%) Remark Psoralen 30 Active ingredients Pull open powder BX 4 Wetting Agents Sodium methylnaphthalene sulfonate formaldehyde condensation 1.5 First dispersant Sodium lignin sulfonate 3 Second dispersant White Carbon Black 3 filler Kaolin Make up 100 filler

[0074] The active ingredient, wetting agent, dispersant and filler are weighed according to the proportion, added into a food processor and mixed evenly, and then put into a jet mill for grinding to obtain the product.

[0075] Field efficacy example: Field trial for controlling citrus canker

[0076] In 2024, field trials were conducted in Wuming County, Guangxi Zhuang Autonomous Region using the drugs prepared in Formulation Examples 2 and 5 to prevent and control citrus canker to further verify the prevention and control effects.

[0077] Test drugs: Preparation Example 2: 25% bakuchiol emulsifiable concentrate; Preparation Example 5: 20% psoralen suspension.

[0078] Control agent: 20% thiophanate-methyl suspension, 500 times diluted, registration number PD20086024, Zhejiang Longwan Chemical Co., Ltd.; blank control: clean water.

[0079] The test method refers to: "GBT17980.103-2004 Guidelines for Field Efficacy Tests of Pesticides (II) Fungicides for the Control of Citrus Canker". Randomized block groups were used, with 4 replicates per treatment and 4 test trees per plot. The first spray was conducted 14 days after the flowers faded, and the second and third sprays were conducted 10 days apart. The pesticide was applied with an electric sprayer to ensure that there was no rain for 24 hours after each application. If it rained, additional spraying should be done in time. The leaves and fruits were sprayed evenly with the pesticide until the liquid was about to drip. The survey began 20 days after the last application of the pesticide. The diagonal sampling method was used. Two citrus trees were surveyed at each point. Each tree was sampled at 5 points in the east, south, west, north, and center. The new leaves on the tender shoots were surveyed at each point, and a total of 200 leaves were surveyed. The number of diseased leaves at each level was recorded and the diseased leaves were graded to calculate the disease index and prevention effect. Taking leaves as the unit, the disease grading standards are as follows

[0080] The grade of citrus canker is divided according to the number of lesions on a single leaf. The grading standards are as follows:

[0081] Level 0: no disease;

[0082] Level 1: 1-5 lesions on each leaf;

[0083] Level 3: 6-10 lesions on each leaf;

[0084] Level 5: 11-15 lesions on each leaf;

[0085] Level 7: 16-20 lesions on each leaf;

[0086] Level 9: There are more than 21 lesions on each leaf.

[0087] The disease index and prevention efficacy were calculated according to formulas (1)-(2):

[0088] Disease index (%) = [∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value)] × 100.

[0089] Relative control effect (%) = (disease index of blank control area after application - disease index of treated area after application) / disease index of blank control area after application × 100.

[0090] Table 3 Results of field efficacy test on the control of citrus canker

[0091]

[0092] It can be seen from the above table that the efficacy of bakuchiol emulsifiable concentrate and psoralen suspension in preventing and controlling citrus canker is significantly higher than that of 20% thiophanate-methyl suspension on the market. Therefore, bakuchiol and psoralen have good application prospects in preventing and controlling citrus canker.

[0093] In summary, among the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia tested in the present invention, bakuchiol, psoralen A and psoralen B have good anti-agricultural pathogenic bacteria activity, especially bakuchiol and psoralen B have excellent antibacterial effects. These compounds have the potential for further development and are expected to become new agricultural antibacterial agents.

[0094] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. Use of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia in the prevention and treatment of agricultural pathogenic bacteria.

2. The use of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia in preventing and treating agricultural pathogenic bacteria according to claim 1, characterized in that: The main secondary metabolites of the traditional Chinese medicine psoralea corylifolia include: psoralen, bakuchiol, psoralen A and psoralen B.

3. The use of the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia in preventing and treating agricultural pathogenic bacteria according to claim 1, characterized in that: The agricultural pathogenic bacteria include: rice bacterial blight pathogen ACCC 11602, citrus canker pathogen, Ralstonia solanacearum, Pseudomonas solanacearum, Xanthomonas campestris and Pseudomonas syringae.

4. A medicament for preventing and treating plant bacterial diseases, characterized in that: The medicament contains the main secondary metabolites of the traditional Chinese medicine Psoralea corylifolia as claimed in claim 2.

5. The agent for preventing and controlling plant bacterial diseases according to claim 4, characterized in that: The weight percentage of the main secondary metabolites of the traditional Chinese medicine psoralea corylifolia in the medicament is 1-99%.

6. The agent for preventing and controlling plant bacterial diseases according to claim 4, characterized in that: The medicament further contains an adjuvant or an agriculturally acceptable carrier.

7. The agent for preventing and controlling plant bacterial diseases according to claim 4, characterized in that: The dosage form of the medicament is a suspension, a dispersible oil suspension, a dry suspension, a wettable powder, an emulsifiable concentrate or a water-dispersible granule.

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