A polyketide compound, its preparation method and application

Abstract

The present invention relates to the fields of microbial medicine technology and natural medicine chemistry, and specifically relates to a polyketide compound obtained from the fermentation product of the fungus Fusarium asiaticum, its separation and purification method, and its application in the activity against aquatic pathogenic bacteria. The molecular formulas of the polyketide compounds are C 29 H 46 O 4 (Compound 1) and C 27 H 38 O 5 (Compound 2), as shown by 1 and 2 in Formula I. The antibacterial activity test shows that Compound 1 and Compound 2 have broad-spectrum antibacterial activities. Among them, Compound 1 has significant inhibitory activities against the aquatic pathogenic bacteria Vibrio parahaemolyticus and Vibrio vulnificus, and their minimum inhibitory concentrations (MICs) are both 1.0 μg / mL, and it is expected to be developed into a new drug for the prevention and treatment of aquatic pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to the fields of microbial medicine technology and natural medicine chemistry, and specifically relates to a polyketide compound obtained from the fermentation product of the fungus Fusarium asiaticum, a method for its isolation, purification and preparation, and its application in anti-aquatic pathogenic bacteria. Background Art

[0002] The fusarin polyketide compounds with a naphthalene ring skeleton structure are a class of bioactive compounds mainly produced by fungi of the genus Fusarium. Their structure contains a naphthalene ring and a three-membered oxygen ring. According to literature research, there is no report on their activity against aquatic pathogenic bacteria.

[0003] Fusarium asiaticum has been documented in the literature and can be isolated from wheat (Zhou Yongjin et al., Jiangsu Journal of Agricultural Sciences, 2012, 5, 979 - 985) and corn (A. Kawakami et al, Journal of General Plant Pathology, 2015, 81(4), 324 - 327). However, so far, there are few systematic research reports on the secondary metabolites of Fusarium asiaticum. The present invention first isolates a polyketide compound with anti-aquatic pathogenic bacteria activity from this fungus. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for obtaining a polyketide compound from the fermentation product of the fungus Fusarium asiaticum, a method for its isolation, purification and preparation, and its application in anti-aquatic pathogenic bacteria.

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

[0006] A polyketide compound, the polyketide compound is compound 1 or compound 2 shown in formula I, wherein the molecular formula of compound 1 is C 29 H 46 O 4 and the molecular formula of compound 2 is C 27 H 38 O 5 ;

[0007]

[0008] A preparation method of a polyketide compound:

[0009] 1) Ferment Fusarium asiaticum in a solid rice medium. The fermentation product is repeatedly soaked and extracted with ethyl acetate. The extracted solutions are combined and concentrated to obtain a crude fermentation extract;

[0010] 2) Subject the crude extract to silica gel column chromatography under reduced pressure, and perform gradient elution successively using petroleum ether - ethyl acetate with a gradient of 20:1 to 1:1 (v / v) and dichloromethane - methanol with a gradient of 20:1 to 1:1 (v / v) as solvents. Collect the fraction eluted with petroleum ether - ethyl acetate at 2:1, and perform RP - 18 reversed - phase silica gel column chromatography, eluting with methanol - water in a ratio of 10:90 to 100:0;

[0011] 3) Collect the fraction of methanol - water at 70:30 from step 2) above, perform normal - phase silica gel column chromatography, elute with dichloromethane - methanol in a ratio of 200:1 to 60:1, collect the fraction of dichloromethane - methanol at 100:1, and then separate and purify it by preparative TLC (the developing agent is petroleum ether: ethyl acetate: acetic acid = 20:10:0.2) to obtain the purified target compound 1;

[0012] 4) Collect the fraction of methanol - water at 60:40 from step 2) above, perform normal - phase silica gel column chromatography, elute with dichloromethane - methanol in a ratio of 80:1 to 10:1, collect the fraction of dichloromethane - methanol at 30:1, and then purify it successively by preparative TLC (the developing agent is dichloromethane: acetone = 10:1) and Sephadex LH - 20 (methanol) to obtain the purified target compound 2;

[0013] Furthermore:

[0014] 1) Inoculate Fusarium asiaticum (sized 2.5 cm × 2.5 cm) grown on a plate medium into a sterilized solid medium and statically culture it at room temperature for 30 days. The fermentation product is repeatedly soaked and extracted with ethyl acetate. The extracted solutions are combined and concentrated to obtain a crude fermentation extract;

[0015] The growth characteristics of Fusarium asiaticum are as follows: on potato sucrose agar (PDA) medium, it has white aerial mycelia, and the mycelial membrane turns pink later; this strain has been reported in many literatures and can be obtained through circulation in multiple preservation centers, such as China Forestry Culture Collection Center (CFCC, preservation number CFCC 51349) and Agricultural Culture Collection of China (ACCC, preservation number ACCC 39255). In addition, those skilled in the art can conveniently isolate it from wheat (Zhou Yongjin et al., Jiangsu Journal of Agricultural Sciences, 2012, 5, 979 - 985) and corn (A. Kawakami et al, Journal of General Plant Pathology, 2015, 81(4), 324 - 327) according to the methods reported in the literature, and can conveniently obtain the registration information of the ITS gene of the strain in the gene bank, for example, NCBI (MK791240.1), NCBI (OM100564.1), etc.

[0016] 2) Subject the crude extract of the fermentation product to reduced pressure silica gel column chromatography, and use petroleum ether - ethyl acetate with a gradient of 20:1 to 1:1 (v / v) and dichloromethane - methanol with a gradient of 20:1 to 1:1 (v / v) as solvents for gradient elution in sequence. Collect the fraction eluted with petroleum ether - ethyl acetate 2:1, and perform RP - 18 reverse - phase silica gel column chromatography, eluting with methanol - water from 10:90 to 100:0;

[0017] 3) Collect the fraction of methanol - water 70:30 in the above step 2), perform normal - phase silica gel column chromatography, elute with dichloromethane - methanol from 200:1 to 60:1, collect the fraction of dichloromethane - methanol 100:1, and then separate and purify it by preparative TLC (developing agent: petroleum ether: ethyl acetate: acetic acid = 20:10:0.2) to obtain the purified target compound 1;

[0018] 4) Collect the fraction of methanol - water 60:40 in the above step 2), perform normal - phase silica gel column chromatography, elute with dichloromethane - methanol from 80:1 to 10:1, collect the fraction of dichloromethane - methanol 30:1, and then purify it successively by preparative TLC (developing agent: dichloromethane: acetone = 10:1) and Sephadex LH - 20 (methanol) to obtain the purified target compound 2.

[0019] The formula of the rice solid medium is: 70 grams of rice, 0.2 grams of corn steep liquor, and 0.3 grams of peptone per 100 milliliters of distilled water.

[0020] Application of a polyketide compound, the application of the polyketide compound shown in Formula I in combating aquatic pathogenic bacteria.

[0021] Application of the polyketide compound shown in Formula I in preparing a new drug for preventing and treating aquatic pathogenic bacteria.

[0022] The aquatic pathogenic bacteria are Aeromonas hydrophila, Edwardsiella ictarda, Edwardsiella tarda, Micrococcus luteus, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahemolyticus or Vibrio vulnificus.

[0023] Advantages of the present invention:

[0024] The present invention uses the fungus Fusarium asiaticum of the genus Fusarium for its fermentation culture. Through optimizing the culture conditions, two new polyketide compounds with significant inhibitory activities against aquatic pathogenic bacteria are obtained from the fermentation products of a rice solid medium. There are currently no reports on the chemical structures and anti-aquatic pathogenic bacteria activities of these compounds, and there are also no related drugs on the market.

[0025] Through the inhibitory activity test of aquatic pathogenic bacteria, Compound 1 and Compound 2 have significant inhibitory activities against aquatic pathogenic bacteria. In particular, Compound 1 has significant inhibitory activities against Vibrio parahemolyticus and Vibrio vulnificus among aquatic pathogenic bacteria, and can be used to prepare drugs for combating aquatic pathogenic bacteria. Specific embodiments

[0026] The following specific examples are used to further illustrate the present invention, but the present invention is by no means limited to these examples.

[0027] The compounds referred to in the following examples are isolated from the fungus Fusarium asiaticum of the genus Fusarium in the present invention. The chemical structures of the compounds are as follows (the Arabic numerals in the structural formula are the carbon atom positions in the chemical structure):

[0028]

[0029] The growth characteristics of the fungus Fusarium asiaticum in the genus Fusarium are that white aerial hyphae grow on potato sucrose agar (PDA) medium, and the mycelial membrane turns pink in the later stage.

[0030] Example 1. Fermentation production, separation and purification of Compounds 1 and 2 shown in Formula I:

[0031] Take the fungus Fusarium asiaticum (size 2.5 cm × 2.5 cm) grown on the plate medium and inoculate it into the sterilized solid medium, and incubate it statically at room temperature for 30 days. The fermentation product is soaked and extracted 3 times with ethyl acetate, and the extraction solutions are combined and concentrated to obtain a crude fermentation extract;

[0032] The formula of the rice solid medium is: 70 grams of rice, 0.2 grams of corn steep liquor, and 0.3 grams of peptone per 100 ml of distilled water.

[0033] The crude extract is subjected to column chromatography on silica gel (100 - 200 mesh) under reduced pressure (the inner diameter of the silica gel column is 10 cm), and gradient elution is carried out successively with petroleum ether - ethyl acetate with a gradient of 20:1 to 1:1 (v / v) and dichloromethane - methanol with a gradient of 20:1 to 1:1 (v / v) as solvents;

[0034] Collect the fraction eluted with petroleum ether - ethyl acetate 2:1, and carry out RP - 18 reverse - phase silica gel column chromatography, eluting with methanol - water from 10:90 to 100:0; collect the fraction of methanol - water 70:30, carry out normal - phase silica gel column chromatography, eluting with dichloromethane - methanol from 200:1 to 60:1, collect the fraction of dichloromethane - methanol 100:1, and then separate and purify it by preparative TLC (the developing agent is petroleum ether: ethyl acetate: acetic acid = 20:10:0.2) to obtain the purified target compound 1;

[0035] Collect the above - mentioned fraction of methanol - water 60:40, carry out normal - phase silica gel column chromatography, eluting with dichloromethane - methanol from 80:1 to 10:1, collect the fraction of dichloromethane - methanol 30:1, and then purify it successively by preparative TLC (the developing agent is dichloromethane: acetone = 10:1) and Sephadex LH - 20 (methanol) to obtain the purified target compound 2; its structure is identified as shown in Formula I,

[0036]

[0037] The two compounds have the following physical and chemical and spectroscopic properties:

[0038] Compound 1: Colorless crystal, mp 135–137 °C; (c 0.49, MeOH); UV(MeOH)λ max(logε)242(3.36)nm; ECD(0.49 mM, MeOH)λ max (Δε)240(–5.58)nm; The 1H and 13C NMR spectra are shown in Table I; HR-EI-MS m / z 458.3392 [M] + , C 29 H 46 O 4 Calcd for 458.3396.

[0039] Compound 2: White amorphous powder, (c 0.21, MeOH); UV (MeOH)λ max (logε)242(3.39)nm; ECD(0.79 mM, MeOH)λ max (Δε)213(–4.99), 246(–2.08)nm; The 1H and 13C NMR spectra are shown in Table I; HR-ESI-MS m / z 441.2635 [M–H] – , C 27 H 37 O 5 Calcd for 441.2646.

[0040] Table I 1H NMR (500 MHz) and 13C NMR (125 MHz) data of compounds 1 and 2 (solvent DMSO-d 6 )

[0041]

[0042] Example 2. Activity of inhibitors against aquatic pathogenic bacteria.

[0043] The antibacterial activities of compounds 1 and 2 of formula I were tested by the minimum inhibitory concentration method. Nine aquatic pathogenic strains were selected: Aeromonas hydrophila, Edwardsiella ictaluri, Edwardsiella tarda, Micrococcus luteus, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus or Vibrio vulnificus for the antibacterial activity test against aquatic pathogenic bacteria.

[0044] 1) Antibacterial activity test (MIC method):

[0045] Minimum inhibitory concentration (MIC), that is, the lowest drug concentration that can inhibit bacterial growth in vitro. In a 96-well microplate, by adding different concentrations of the drug to the bacterial suspension of the test bacterium and observing after incubation, if the indicator bacterium grows in a certain well, it means that the drug concentration in that well cannot inhibit the growth of the bacterium, and the liquid in that well is turbid and the light transmittance decreases significantly. On the contrary, the liquid in that well is clear and the decrease in light transmittance is not significant. The lowest sample concentration that completely inhibits the growth of the indicator bacterium in the small well is the MIC of the compound.

[0046] 2) Preparation of bacterial suspension

[0047] The above-mentioned test bacteria were respectively inoculated on the culture medium (for Aeromonas hydrophila A. hydrophila, Edwardsiella ictaluri E. ictarda, Micrococcus luteus M. luteus, Vibrio harveyi V. harveyi, Vibrio parahaemolyticus V. Parahemolyticus, LB medium was used; for Edwardsiella tarda E. tarda, Pseudomonas aeruginosa P. aeruginosa, Vibrio alginolyticus V. alginolyticus and Vibrio vulnificus V. vulnificus, TSB medium was used), cultured at 28 °C for 24 hours, then an appropriate amount of the bacterial suspension was aspirated into a sterile test tube with a pipette gun, and then the bacterial suspension was adjusted to 0.5 McFarland turbidity (equivalent to 1.5×10 8 CFU / mL) with 0.85% NaCl solution, and further diluted to 5×10 5 CFU / mL with 0.85% NaCl solution.

[0048] 0.5 McFarland turbidity standard:

[0049] Add 0.5 mL of 0.048 mol / L BaCl 2 (1.175% w / v BaCl 2 ·2H 2 O) to 99.5 mL of 0.18 mol / L (0.36 N) H 2 SO 4 (1% v / v) and stir continuously to maintain the suspension state.

[0050] 3) Preparation of samples

[0051] Weigh approximately 1 mg of the sample to be tested (Compound 1 or Compound 2 obtained above), dissolve it in approximately 100 μL of DMSO, mix well, and make the final concentration 2560 μg / mL. Pipette 50 μL of the sample solution into another centrifuge tube, and then add 50 μL of DMSO to obtain a sample solution with a halved concentration. Using this method, a total of 11 sets of sample solutions with successively halved concentrations (2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 μg / mL) are obtained.

[0052] 4) Blank control: Select the pure solvent (DMSO) for dissolving the sample to be tested as the blank control.

[0053] 5) MIC determination procedure

[0054] 5.1) Using aseptic technique, add the serially diluted sample solutions with different concentrations to a sterile 96-well plate. Add the sample solution to wells 1 to 11, 5 μL per well, and do not add anything to well 12 as the growth control.

[0055] 5.2) Dilute the indicator bacteria suspension equivalent to 0.5 McFarland turbidity 1000-fold with a liquid medium (for Aeromonas hydrophila A. hydrophila, Edwardsiella ictaluri E. ictarda, Micrococcus luteus M. luteus, Vibrio harveyi V. harveyi, Vibrio parahaemolyticus V. Parahemolyticus, use LB medium; for Edwardsiella tarda E. tarda, Pseudomonas aeruginosa P. aeruginosa, Vibrio alginolyticus V. alginolyticus, and Vibrio vulnificus V. vulnificus, use TSB medium), and then take 95 μL and add it to the 96-well plate in sequence, so that the final concentrations of the samples in wells 1 to 11 are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL respectively. After gently shaking and mixing, seal the 96-well plate and place it in an incubator at 28 °C for 24 h of bacterial culture.

[0056] 5.3) Use a microplate reader to measure the absorbance value of each well at a wavelength of 600 nm, and take the lowest sample concentration that completely inhibits the growth of the indicator bacteria in the well as the MIC of the compound. (Note: The experiment is meaningful only when the indicator bacteria in the negative control well grow significantly; when there is a single skipped well in the experiment, record the highest drug concentration that inhibits the growth of the strain; if there are multiple skipped wells, the result should not be reported and the experiment needs to be repeated.)

[0057] Table II Antibacterial activity data of Compound 1 (MIC, μg / mL)

[0058] Test bacteria AH ML PA VA VH VP VV Compound 1 4 2 2 4 4 1 1

[0059] Antibacterial activity data (MIC, μg / mL) of Compound 2 in Table III

[0060] Test bacteria AH ML PA VP VV Compound 2 2 4 4 4 2

[0061] Note: In Table II and Table III above, AH: Aeromonas hydrophila; ML: Micrococcus luteus; PA: Pseudomonas aeruginosa; VA: Vibrio alginolyticus; VH: Vibrio harveyi; VP: Vibrio parahaemolyticus; VV: Vibrio vulnificus.

[0062] The experimental results show that Compounds 1 and 2 have good and broad-spectrum antibacterial activities. As shown in Table II and Table III, in the antibacterial activity experiment, Compounds 1 and 2 have strong inhibitory activities against Aeromonas hydrophila, Micrococcus luteus, Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio vulnificus, and the MIC values are 1–4 μg / mL. Compound 1 also has strong inhibitory activities against Vibrio alginolyticus and Vibrio harveyi, and the MIC values are both 4 μg / mL.

[0063] The above experimental results prove that the compounds involved in the present invention have strong inhibitory effects on aquatic pathogenic bacteria, and they can be used to prepare new drugs for preventing and treating aquatic pathogenic bacteria.

Claims

1. A polyketide compound, characterized in that: The polyketide compound is compound 1 or compound 2 shown in formula I, wherein the molecular formula of compound 1 is C 29 H 46 O 4、 The molecular formula of compound 2 is C 27 H 38 O 5 ; Formula I.

2. A method for preparing the polyketide compound according to claim 1, characterized in that: 1) Ferment the fungus Fusarium asiaticum Fusarium asiaticum in a solid rice medium. The fermentation product is repeatedly soaked and extracted with ethyl acetate, and the extraction liquids are combined and concentrated to obtain a crude fermentation extract. 2) The crude extract is subjected to silica gel column chromatography under reduced pressure, and gradient elution is carried out successively with petroleum ether-ethyl acetate with a gradient of 20:1 to 1:1 by volume ratio and dichloromethane-methanol with a gradient of 20:1 to 1:1 by volume ratio as solvents. The fractions eluted with petroleum ether-ethyl acetate 2:1 are collected and subjected to RP-18 reversed-phase silica gel column chromatography, and eluted with methanol-water with a ratio of 10:90 to 100:0; 3) The fractions of methanol-water 70:30 in the above step 2 are collected, subjected to normal-phase silica gel column chromatography, and eluted with dichloromethane-methanol with a ratio of 200:1 to 60:

1. The fractions of dichloromethane-methanol 100:1 are collected and then separated and purified by preparative TLC. Among them, the developing agent is petroleum ether: ethyl acetate: acetic acid = 20:10:0.2 to obtain the purified target compound 1; 4) The fractions of methanol-water 60:40 in the above step 2 are collected, subjected to normal-phase silica gel column chromatography, and eluted with dichloromethane-methanol with a ratio of 80:1 to 10:

1. The fractions of dichloromethane-methanol 30:1 are collected and then separated and purified successively by preparative TLC and Sephadex LH-20 methanol. Among them, the developing agent for preparative TLC is dichloromethane: acetone = 10:1 to obtain the purified target compound 2.

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

4. An application of compound 1 shown in Formula I in claim 1, characterized in that: Use of the compound in the preparation of a drug for preventing and treating aquatic pathogenic bacteria; the aquatic pathogenic bacteria are Aeromonas hydrophila Aeromonas hydrophila , Micrococcus luteus Micrococcus luteus , Pseudomonas aeruginosa Pseudomonas aeruginosa , Vibrio alginolyticus Vibrio alginolyticus , Vibrio harveyi V. harveyi , Vibrio parahaemolyticus V. Parahemolyticus or Vibrio vulnificus V. vulnificus .

5. An application of compound 2 shown in Formula I in claim 1, characterized in that: Use of the compound in the preparation of a drug for preventing and treating aquatic pathogenic bacteria; the aquatic pathogenic bacteria are Aeromonas hydrophila Aeromonas hydrophila , Micrococcus luteus Micrococcus luteus , Pseudomonas aeruginosa Pseudomonas aeruginosa , Vibrio parahaemolyticus V. Parahemolyticus or Vibrio vulnificus V. vulnificus .

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