Use of a berberine derivative in the control of brown rot in stone fruits

Abstract

This invention discloses the use of any compound among berberine derivatives in controlling diseases caused by postharvest rot-causing fungi in fruits, belonging to the field of medicinal chemistry. These derivatives exhibit excellent inhibitory activity against *Monilinia fructicola* ACCC 36263, the causal agent of brown rot in stone fruits, present in food. The compounds involved in this invention have a simple preparation process, use inexpensive and readily available raw materials, and hold promise for development into a novel food preservative and antifungal agent.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and discloses a new use of berberine derivatives, specifically involving the use of berberine derivatives A1-A37, D1-D7, E1-E7, F1-F5 and Z1-Z3 in the prevention and control of brown rot caused by the drupe fungus ACCC 36263. Background Technology

[0002] Postharvest rot of fresh fruit has become a widespread problem globally. This is primarily due to three factors: physiological imbalances or aging of fruit tissues, pathogenic microbial infection, and postharvest mechanical damage. These three factors interact, with pathogenic microorganisms being the most significant and ultimately the cause of rot. Fungal infections account for the largest proportion and cause the most damage, resulting in severe economic losses and harming human health. Examples include Penicillium, Botrytis, and brown rot of stone fruits.

[0003] The Australian-American type of brown rot fungus, also known as stone fruit brown rot fungus, is an infectious rot-causing fungus that causes brown rot in postharvest stone fruits, causing particularly severe damage to peaches, plums, and apricots. The pathogen initially appears as dark spots on the fruit surface, which then develop into dark brown lesions producing numerous grayish-brown conidia, causing the fruit to rot or shrivel into mummified fruit. Currently, the control of this fungus mainly relies on chemical control, such as with azoxystrobin and thiophanate-methyl. However, long-term use leads to problems such as pesticide residues, resistance, and environmental pollution. Therefore, researching and developing new, highly effective, low-toxicity, low-residue, and environmentally safe agricultural fungicides is one of the main goals of pesticide development.

[0004] Natural products possess abundant biological activity and low toxicity, serving as a primary source of inspiration for new drug development. Compared to traditional synthetic fungicides, these compounds based on natural product sources exhibit lower toxicity and environmental compatibility, making them worthy of in-depth utilization and development. Berberine, a quaternary ammonium isoquinoline alkaloid, was first isolated from the bark of *Xanthoxylon clava*. Current reports on the biological activities of this alkaloid and its derivatives mainly focus on antibacterial, anti-inflammatory, and antitumor effects, while reports on their application in postharvest disease control of fruit, particularly brown rot, are extremely limited. Therefore, the berberine derivative described in this invention exhibits significant antibacterial activity against *ACCC 36263*, the causal agent of brown rot in stone fruits, and is significantly superior to the control drug, protoberberine, suggesting its potential for further development as a novel agricultural fungicide. Summary of the Invention

[0005] The purpose of this invention is to provide a new use of berberine derivatives in the prevention and control of brown rot caused by the drupe causal agent ACCC 36263.

[0006] To achieve the above objectives, the present invention provides the following technical method:

[0007] This invention proposes a novel use of berberine derivatives in the prevention and control of brown rot caused by ACCC 36263, the causal agent of brown rot in stone fruits. The dosage of these derivatives against the causal agent of brown rot in stone fruits is 50, 25, 10, 5, 2.5, 1, 0.5, and 0.25 μg / mL.

[0008] Furthermore, the berberine derivatives described in this invention were synthesized using methods reported in the literature. Pure products were obtained by separation using conventional methods such as multiple silica gel column chromatography. The structures of berberine derivatives A1-A37, D1-D7, E1-E7, F1-F5, and Z1-Z3 were determined by spectroscopic techniques such as mass spectrometry and nuclear magnetic resonance. Berberine derivatives A1 to A37 are shown in chemical formula 1, wherein R1, R2, R3, R4, and R5 can be hydrogen, fluorine, chlorine, bromine, iodine, cyano, methoxy, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, tert-butyl, isopropyl, or methyl; berberine derivatives D1 to D7 are shown in chemical formula 2; berberine derivatives E1 to E7 are shown in chemical formula 3, wherein n is 0 to 8 carbon atoms; berberine derivatives F1 to F5 are shown in chemical formula 4, wherein R6 is an alkane or a benzene ring, etc.; berberine derivatives Z1 to Z3 are shown in chemical formula 5.

[0009]

[0010] Specifically, the chemical structural formula of the berberine derivative is as follows:

[0011]

[0012]

[0013]

[0014] The bactericide provided by this invention has the following advantages:

[0015] 1) This invention found that berberine derivatives exhibit excellent inhibitory effects on ACCC 36263, the causal agent of brown rot in stone fruits, by inhibiting the formation and growth of fungal mycelium.

[0016] 2) This berberine derivative has the characteristics of simple structure, easy synthesis, environmental compatibility, and low resistance. It can be used as an effective ingredient in the field of food anti-mold and anti-corrosion, and has the potential to be developed into a new type of fruit and vegetable preservative. Detailed Implementation

[0017] To better understand the present invention, the following specific embodiments further illustrate the above-described content of the present invention. However, this should not be construed as a limitation of the present invention. The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention. However, the content of the present invention is not limited to the scope described in the embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0018] Example 1: Activity assay of berberine derivatives against ACCC 36263, the pathogen causing brown rot of stone fruit.

[0019] 1) Test reagent: Berberine derivative.

[0020] 2) Test strain: Monilinia fructicola (ACCC 36263), the causal agent of brown rot of stone fruit, was provided by Gansu Academy of Agricultural Sciences.

[0021] 3) Testing methods:

[0022] The mycelial growth rate method was used to determine the activity of the mycelium in the laboratory. The culture medium was potato agar-glucose medium (PDA). The PDA medium formula was: 200g potato (peeled), 20g glucose, 15g agar, and 1000mL distilled water.

[0023] PDA culture medium preparation method: Wash and peel potatoes, weigh 200g and cut into small pieces. Boil in distilled water for about 20 minutes (until the potato pieces are soft but not mushy). Filter through eight layers of gauze. Add distilled water to make up to 1000mL of the filtrate. Add 15g of agar and 20g of glucose. Stir to dissolve completely. Dispense into Erlenmeyer flasks and sterilize at 121℃ for 20 minutes. Cool before use.

[0024] Strain activation: Incubate the brown rot pathogen of stone fruit on PDA plates at 25°C for 3–6 days.

[0025] Preparation of drug-coated plates: Cool the PDA medium sterilized at 121℃ to 45-50℃, and add the test reagent to make drug-coated plates with concentrations of 500, 250, 100, 50, 25, 10, and 5 μg / mL respectively.

[0026] Inoculation and cultivation: In a clean bench, use a punch to collect mycelial cakes (5 mm in diameter) from the edge of the mycelium after 3-6 days of cultivation (with the growth conditions as uniform as possible), then use an inoculation needle to pick them up and transfer them to the center of the substrate. Finally, incubate the substrate upside down in an incubator (28℃).

[0027] Results: After the hyphae in the blank control group had fully grown, the growth diameter of the hyphae in the drug-treated group was measured using the cross-cross method, and the percentages of growth inhibition (PGI) were calculated.

[0028] Mycelial growth inhibition rate (%) = (control mycelial diameter - treated mycelial diameter) / (control mycelial diameter - mycelial cake diameter) × 100. Three parallel experiments were set up for each concentration. The inhibition rate of the compound was measured and is shown in Table 1.

[0029] Table 1. Inhibition rate (%) of berberine derivatives against brown rot pathogen of stone fruit at 50 μg / mL

[0030]

[0031]

[0032] The above results indicate that the berberine derivatives involved in this invention exhibit excellent antifungal effects against *Pseudomonas aeruginosa*, the causal agent of brown rot in stone fruits. Compounds with an inhibition rate greater than 50% at 50 μg / mL were selected, and their inhibition rates were further tested at lower concentrations, including 25, 10, 5, 2.5, 1, 0.5, and 0.25 μg / mL. The half-maximal effective concentration (EC50) was then determined using SPSS software. 50 The activity data obtained are shown in Table 2.

[0033] Table 2. EC50 of berberine derivatives against *Pseudomonas aeruginosa*, the causal agent of brown rot in stone fruits. 50 Value (μg / mL)

[0034]

[0035]

[0036] As shown in Tables 1 and 2, the berberine derivatives involved in this invention exhibit good fungicidal activity against ACCC 36263, the causal agent of brown rot in stone fruits, with inhibition rates mostly greater than 95% at 50 μg / mL. The EC50 of most derivatives... 50 The values ​​were significantly better than those of berberine and pyrimethanil. Among them, A17, A31, A37, and D2 showed the best activity, with EC... 50 The value is <1 μg / mL, which is superior to the positive control drug natamycin.

[0037] In summary, the berberine derivatives described in this invention exhibit good antibacterial activity against ACCC 36263, the causal agent of brown rot in stone fruits, and have further research and development value in food preservation.

Claims

1. Use of a berberine derivative in the control of Monilinia fructicola ACCC 36263, wherein, The berberine derivative has the following molecular structural characteristics:

Images