A polyketide compound, a preparation method thereof and application thereof in resisting gram-negative bacteria

Abstract

The present application relates to the field of natural pharmaceutical chemistry and microbial medicine technology, and particularly relates to a novel polyketide compound extracted from a fermentation product of a fungus Fusarium asiaticum, a method for separating and purifying the polyketide compound, and application of the polyketide compound in anti-Gram-negative bacteria activity. 22 H 30 O3 (compound 1) and C 24 H 34 O2 (compound 2), as shown in 1 and 2 in formula I. The compound 1 and the compound 2 exhibit strong anti-Gram-negative bacteria activity. Among them, the compound 1 can significantly inhibit the growth of Gram-negative bacteria Aeromonas hydrophilia, Vibrio alginolyticus and V. harveyi, and the minimum inhibitory concentration (MIC) is 1.0, 2.0 and 2.0 μg / mL respectively; the compound 2 can significantly inhibit the growth of Gram-negative bacteria V. harveyi, and the MIC is 2.0 μg / mL. The compound 1 and the compound 2 have the potential to become new Gram-negative bacteria prevention and treatment drugs, and are expected to provide a new direction for solving the problems in the field.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry / microbial pharmaceutical technology, specifically to the extraction of novel polyketide compounds from the fermentation products of the fungus Fusarium asiaticum, its preparation method, and its application in combating Gram-negative bacteria. Background Technology

[0002] Polyketides are a class of natural products with broad biological activities, some of which have been developed and applied as clinical drugs, such as doxorubicin, tetracycline, and erythromycin. With the deepening exploration of microbial resources, polyketide pesticides extracted from actinomycetes, including avermectins, echinocandins, tetracyclines, and their analogues, can effectively protect plants from various insects and parasites and have been successfully commercialized.

[0003] Research on *F. asiaticum* has been documented, and this fungus can be isolated from wheat (Zhou Yongjin et al., *Jiangsu Journal of Agricultural Sciences*, 2012, 5, 979-985) and maize (A. Kawakami et al., *Journal of General Plant Pathology*, 2015, 81(4), 324-327). It is noteworthy that there are relatively few reports on systematic studies of the secondary metabolites of *F. asiaticum*. This invention successfully isolated a novel polyketide compound with anti-Gram-negative bacterial activity from this fungus. Summary of the Invention

[0004] The purpose of this invention is to provide a novel polyketide compound extracted from the fermentation products of the fungus Fusarium asiaticum, a method for its preparation, and its application in combating Gram-negative bacteria.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A polyketide compound, wherein the polyketide compound is as shown in Formula I.

[0007]

[0008] In the formula, compound 1: R = -CHO, compound 2: R = -CH = CHCH3.

[0009] A method for preparing the aforementioned polyketide compound:

[0010] 1) The fungus Fusarium asiaticum was inoculated into rice solid culture medium and fermented at room temperature for 30 days. After the fermentation was completed, the product was repeatedly extracted by ethyl acetate. The extracts were combined and concentrated to obtain the crude fermentation extract.

[0011] 2) The crude extract was separated by vacuum silica gel column chromatography. The crude extract was eluted sequentially with a gradient of 20:1 to 1:1 (v / v) petroleum ether-ethyl acetate and a gradient of 20:1 to 1:1 (v / v) dichloromethane-methanol. The fraction obtained by elution with petroleum ether-ethyl acetate at a ratio of 1:1 was collected and subjected to reversed-phase silica gel (RP-18) column chromatography with methanol-water at a ratio of 10:90 to 100:0.

[0012] 3) Collect the methanol-water 60:40 fraction from step 2) and perform normal-phase silica gel column chromatography, eluting with dichloromethane-methanol at a ratio of 200:1 to 10:1. Collect the dichloromethane-methanol 150:1 fraction and then purify it by preparative TLC to obtain the purified target compound 1 (molecular formula C10) as described in Formula I, R = -CHO. 22 H 30 O3);

[0013] 4) Collect the methanol-water 90:10 fraction from step 2) and purify it using LH-20 gel permeate (methanol) to obtain the purified target compound 2 (molecular formula C) as described in Formula I, R=-CH=CHCH3. 24 H 34 O2).

[0014] Specifically:

[0015] 1) The fungus *Fusarium asiaticum* was streaked onto PDA medium and incubated at 28°C for 7 days. A 2.5 cm × 2.5 cm mycelial block was then inoculated into sterilized rice solid medium, ensuring full contact between the mycelial block and the medium. After inoculation, the mixture was incubated statically at room temperature for 30 days to obtain the fermentation product. The fermentation product was repeatedly extracted with ethyl acetate, and the extracts were combined and concentrated under reduced pressure to obtain the crude fermentation extract.

[0016] The *F. asiaticum* strain is characterized by white, fluffy aerial mycelia growing on potato sucrose agar (PDA) medium. Initial growth is slow, but it gradually spreads, forming dense colonies, and the mycelial film later turns pink. This strain has been reported in several publications and can be purchased through the Agricultural Culture Collection of China (ACCC, accession number ACCC 39255) via [http: / / accc.org.cn / Column.asp?Column_ID=34929&Model=product_detail&P_ID=10214248].

[0017] 2) The crude extract of the fermentation product was subjected to reduced pressure silica gel column chromatography, and the fraction was eluted sequentially with a gradient of 20:1 to 1:1 (v / v) petroleum ether-ethyl acetate and a gradient of 20:1 to 1:1 (v / v) dichloromethane-methanol. The fraction obtained by elution with petroleum ether-ethyl acetate 1:1 was collected and subjected to reversed phase silica gel column (RP-18) chromatography, eluted with methanol-water at a ratio of 10:90 to 100:0.

[0018] 3) Collect the elution fraction of methanol-water 60:40 from step 2) above, perform normal phase silica gel column chromatography, elute with dichloromethane-methanol at a ratio of 200:1 to 10:1, collect the elution fraction of dichloromethane-methanol 150:1, and perform preparative TLC (petroleum ether: ethyl acetate = 1:1 as the developing solvent) to obtain purified target compound 1;

[0019] 4) Collect the elution fraction of methanol-water 90:10 from step 2) above, and purify it by gel LH-20 (methanol) to obtain purified target compound 2.

[0020] The rice solid culture medium formula is as follows: 70 grams of rice, 0.2 grams of corn steep liquor, and 0.3 grams of peptone per 100 ml of distilled water.

[0021] An application of the polyketide compound described herein, specifically the application of the polyketide compound of Formula I in the inhibition of Gram-negative bacteria.

[0022] The application of the polyketide compounds shown in Formula I in the preparation of drugs for the prevention and treatment of Gram-negative pathogens.

[0023] The Gram-negative pathogens mentioned are Aeromonas hydrophilia, Edwardsiella ictarda, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harvesti, Vibrio parahemolyticus, or Vibrio vulnificus.

[0024] Advantages of this invention:

[0025] This invention utilizes the fungus *F. asiaticum* for fermentation culture. Through optimized culture conditions, two novel polyketide compounds were isolated from the fermentation products on a rice solid medium. To date, no reports have been found regarding the chemical structure or activity against Gram-negative pathogens of these two compounds. Compared to normal fusarinin, compound 2 lacks a carbon atom in its polyene side chain, possibly due to the absence of S-adenosine-L-methionine methylation during biosynthesis—an uncommon and previously unreported characteristic.

[0026] The results of the inhibitory activity test on Gram-negative pathogens showed that compounds 1 and 2 had significant inhibitory activity against Gram-negative bacteria. In particular, compound 1 had a significant inhibitory effect on Aeromonas hydrophila, Vibrio alginolyticus and Vibrio harveyi, and compound 2 had a significant inhibitory effect on Vibrio harveyi. They are expected to be used in the preparation and development of a new drug against Gram-negative pathogens. Detailed Implementation

[0027] The following specific embodiments are used to further illustrate the present invention, but the present invention is by no means limited to these examples.

[0028] The present invention isolated the compounds referred to in the following examples from the fungi of the genus *Fusarium*, *F. asiaticum*. The chemical structures of the compounds are as follows (the Arabic numerals in the structural formulas are the carbon atom markers in the chemical structures):

[0029]

[0030] In the formula, compound 1: R = -CHO, compound 2: R = -CH = CHCH3.

[0031] The growth characteristics of the fungus *F. asiaticum* are as follows: white, fluffy aerial hyphae grow on potato sucrose agar (PDA) medium. Initially, the growth is relatively slow, but it gradually spreads and forms dense colonies. Later, the mycelium turns pink.

[0032] Example 1. Fermentation production and purification of compounds 1 and 2 shown in Formula I:

[0033] First, the fungus Fusarium asiaticum (2.5 cm × 2.5 cm in size) grown on PDA plates for 7 days was inoculated into sterilized rice solid culture medium, ensuring full contact between the fungal blocks and the rice culture medium. After inoculation, the mixture was incubated at room temperature for 30 days. The fermentation product was repeatedly extracted by soaking with ethyl acetate. The extracts were combined and concentrated to obtain the crude fermentation extract.

[0034] The rice solid culture medium formula is as follows: 70 grams of rice, 0.2 grams of corn steep liquor, and 0.3 grams of peptone per 100 ml of distilled water.

[0035] The crude extract was subjected to vacuum silica gel (100-200 mesh) column chromatography (silica gel column inner diameter 10 cm), and was eluted sequentially with petroleum ether-ethyl acetate in a gradient of 20:1 to 1:1 (v / v) and dichloromethane-methanol in a gradient of 20:1 to 1:1 (v / v) as solvents.

[0036] The fraction obtained by elution with petroleum ether and ethyl acetate at a 1:1 ratio was collected and subjected to reversed-phase silica gel (RP-18) column chromatography, eluted with methanol-water at a ratio of 10:90 to 100:0; the fraction obtained by elution with methanol-water at a 60:40 ratio was collected and subjected to normal-phase silica gel column chromatography, eluted with dichloromethane-methanol at a ratio of 200:1 to 10:1; the fraction obtained by elution with dichloromethane-methanol at a 150:1 ratio was collected and then purified by preparative TLC (petroleum ether:ethyl acetate = 1:1 as the developing solvent) to obtain purified target compound 1;

[0037] The methanol-water 90:10 fraction was collected and purified using LH-20 gel electrophoresis (methanol) to obtain purified target compound 2; its structure was identified as shown in Formula I.

[0038]

[0039] In the formula, compound 1: R = -CHO, compound 2: R = -CH = CHCH3.

[0040] The two compounds have the following physicochemical and spectroscopic properties:

[0041] Compound 1: Colorless crystals, mp 198-200℃; UV(MeOH)λmax (logε)289(3.48)nm; ECD(0.37mM,MeOH)λ max (Δε) 280(-8.75) nm; 1H and 1C NMR spectra are shown in Table I; High-resolution ESI mass spectra m / z 341.2113 [MH] - C 22 H 29 The calculated value of O3 is 341.2122.

[0042] Compound 2: a colorless oily substance. UV(MeOH)λ max (logε)242(3.04), 275(2.75)nm; ECD(0.85mM, MeOH)λ max (Δε) 283(-3.52) nm; 1H and 1C NMR spectra are shown in Table I; EI mass spectra m / z 354 [M] + C 24 H 34 The calculated value for O2 is 354. Table I shows the 1H NMR (500MHz) and 1C NMR (125MHz) spectra of compounds 1 and 2 (solvent DMSO-d6).

[0043]

[0044] Note: The signal attribution in this table is based on DEPT. 1 H- 1 The multiplicity of the carbon signal was determined using the DEPT method based on the analysis results of the H COSY, HSQC, and HMBC spectra.

[0045] Example 2. Inhibitory activity against Gram-negative bacteria.

[0046] The activity of compounds 1 and 2 of Formula I against Gram-negative bacteria was determined using the minimum inhibitory concentration (MIC) method. Seven Gram-negative pathogenic bacteria were selected for testing their activity against aquatic pathogens: *Aeromonas hydrophila*, *E. ictarda*, *Pseudomonas aeruginosa*, *V. alginolyticus*, *V. harvestyi*, *V. parahemolyticus*, and *V. vulnificus*.

[0047] 1) Antibacterial activity test (MIC method):

[0048] The minimum inhibitory concentration (MIC) is the lowest concentration of a drug that can inhibit the growth of microorganisms in vitro. In a 96-well microplate, different concentrations of the drug are added to a bacterial suspension of the test bacteria. After incubation, the MIC is observed. If indicator bacteria grow in a well, it means the drug concentration in that well is insufficient to inhibit their growth; the liquid in that well will be turbid, and the transmittance will be significantly reduced. Conversely, if the liquid in that well is clear, the transmittance will not decrease significantly. The lowest sample concentration at which the indicator bacteria are completely inhibited within the well is the MIC of the compound.

[0049] 2) Preparation of bacterial suspension

[0050] The tested bacteria were inoculated onto culture media (LB medium for Aeromonas hydrophila, Edwardsiella ictarda, Vibrio harvesti, and Vibrio parahaemolyticus; TSB medium for Pseudomonas aeruginosa, Vibrio alginolyticus, and Vibrio vulnificus) and incubated at 28°C for 24 hours. A suitable amount of bacterial suspension was then pipetted into sterile test tubes, and the suspension was adjusted to 0.5 McFarland turbidity (equivalent to 1.5 × 10⁻⁶) with 0.85% NaCl solution. 8 The concentration was (CFU / mL) and further diluted to 5 × 10⁻⁶ with 0.85% NaCl solution. 5 CFU / mL.

[0051] 0.5 McFarland turbidity standard:

[0052] Add 0.5 mL of 0.048 mol / L BaCl2 (1.175% w / v BaCl2·2H2O) to 99.5 mL of 0.18 mol / L (0.36 N) H2SO4 (1% v / v) and stir continuously to maintain suspension.

[0053] 3) Sample preparation

[0054] Take approximately 1 mg of the test sample (i.e., compound 1 or compound 2 mentioned above), dissolve it in approximately 100 μL of DMSO, mix thoroughly to achieve a final concentration of 2560 μL / mL. Then, transfer 50 μL of this sample solution to another centrifuge tube and add 50 μL of DMSO to obtain a sample solution with half the concentration. Using the same method, 11 groups of sample solutions with sequentially halved concentrations can be obtained, with the concentration gradients being: 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, and 2.5 μg / mL.

[0055] 4) Blank control: The pure solvent (DMSO) used to dissolve the sample to be tested is used as a blank control.

[0056] 5) MIC Measurement Procedure

[0057] 5.1) Sample addition

[0058] Aseptic technique was used to add sample solutions of different concentrations after serial dilution to sterile 96-well plates. 5 μL of sample solution was added to wells 1 to 11, and well 12 was left untreated as a growth control.

[0059] 5.2) Adding bacteria

[0060] A suspension of indicator bacteria with a turbidity equivalent to 0.5 McFarland ratio was diluted 1000-fold with liquid culture medium (LB medium for Aeromonas hydrophila, E. ictarda, V. harvestyi, and V. parahemolyticus; TSB medium for Pseudomonas aeruginosa, V. alginolyticus, and V. vulnificus). 95 μL of each solution was then added sequentially to 96-well plates, resulting in final sample concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL for wells 1 to 11, respectively. After gentle shaking to mix, the 96-well plates were sealed and incubated at 28°C for 24 hours.

[0061] 5.3) Observation of Results

[0062] After culturing, the absorbance of each well at 600 nm was measured using a microplate reader. The lowest sample concentration that completely inhibited the growth of indicator bacteria in the well was defined as the MIC of the compound. (Note: The experiment is only meaningful when the indicator bacteria show obvious growth in the negative control well; when a single well skips a test, the highest drug concentration that inhibits the growth of the strain should be recorded; if multiple wells skip a test, the results should not be reported, and the experiment should be repeated.)

[0063] Table II shows the antibacterial activity data (MIC, μg / mL) of compounds 1 and 2.

[0064]

[0065] The experimental results showed that compounds 1 and 2 had certain antibacterial activities against the seven tested Gram-negative bacteria, as shown in Table II. Compound 1 had strong inhibitory activity against Aeromonas hydrophila, Vibrio alginolyticus, and Vibrio harvestyi, with MIC values ​​of 1-2 μg / mL. Compound 2 had strong inhibitory effects against Vibrio harvestyi and Vibrio vulnificus, with MIC values ​​of 2-4 μg / mL.

[0066] The above experimental results demonstrate that the compounds involved in this invention have a strong inhibitory effect on Gram-negative bacteria, and they can be used to prepare novel drugs for the prevention and treatment of Gram-negative pathogens, which can be applied in aquaculture and other fields.

Claims

1. A polyketide compound, characterized in that: Polyketide compounds are shown in Formula I. Formula I; In the formula, compound 1: R=−CHO, compound 2: R=−CH=CHCH3.

2. A method for preparing the polyketide compound according to claim 1, characterized in that: 1) The fungus Fusarium subspecies ( Fusarium asiaticum The product was inoculated into rice solid culture medium and fermented at room temperature for 30 days. After the fermentation was completed, the product was repeatedly extracted by ethyl acetate. The extracts were combined and concentrated to obtain the crude fermentation extract. 2) The crude extract was separated by vacuum silica gel column chromatography. The crude extract was eluted sequentially with a gradient of petroleum ether-ethyl acetate (v / v) at a ratio of 20:1 to 1:1 and dichloromethane-methanol (v / v) at a ratio of 20:1 to 1:

1. The fraction obtained by elution with petroleum ether-ethyl acetate at a ratio of 1:1 was collected and subjected to RP-18 reversed-phase silica gel column chromatography with methanol-water at a ratio of 10:90 to 100:

0. 3) Collect the methanol-water 60:40 fraction from step 2) and perform normal phase silica gel column chromatography, eluting with dichloromethane-methanol at a ratio of 200:1 to 10:

1. Collect the dichloromethane-methanol 150:1 fraction and then separate and purify it by preparative TLC to obtain 8.1 mg of the purified target compound 1 as described in Formula I, R=−CHO. 4) Collect the methanol-water 90:10 component from step 2) above and purify it using gel LH-20 to obtain 4.1 mg of the purified target compound 2 as described in Formula I, R=−CH=CHCH3.

3. The method for preparing the polyketide compound according to claim 2, characterized in that: The rice solid culture medium formula is as follows: 70 grams of rice, 0.2 grams of corn steep liquor, and 0.3 grams of peptone per 100 ml of distilled water.

4. An application of the polyketide compound according to claim 1, characterized in that: The application of the polyketide compounds shown in Formula I in the preparation of drugs for the prevention and treatment of Gram-negative pathogens; The Gram-negative pathogen is Aeromonas hydrophila. Aeromonas hydrophilia Edwardsiella catfish Edwardsiella ictarda Pseudomonas aeruginosa Pseudomonas aeruginosa Vibrio alginolyticus Vibrio alginolyticus Vibrio harzianum V. harveyi Vibrio parahaemolyticus V. Parahemolyticus or Vibrio vulnificus V. vulnificus .

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