Polyketone compound, preparation method thereof and application of polyketone compound in gram-negative bacterium resistance

Abstract

The invention relates to the technical field of natural medicinal chemistry and microbial medicine, in particular to a method for extracting a novel polyketone compound from a fermentation product of fungus fusarium asiaticum, a method for separating and purifying the novel polyketone compound and application of the novel polyketone compound in the aspect of gram-negative bacterium activity resistance. The molecular formulas of the polyketone compounds are respectively C22H30O3 (compound 1) and C24H34O2 (compound 2), as shown in the formula I and the formula II. The compound 1 and the compound 2 show relatively strong gram-negative bacterium resisting activity. Wherein the compound 1 can be used for remarkably inhibiting the growth of Aeromonas hydrophila, Vibrio alginolyticus and Vibrio harveyi which are gram-negative bacteria, and the minimum inhibitory concentrations (MIC) of the compound 1 are 1.0 mu g / mL, 2.0 mu g / mL and 2.0 mu g / mL in sequence; the compound 2 can significantly inhibit the growth of gram-negative bacterium vibrio harveyi V.harveyi, and the MIC of the compound 2 is 2.0 [mu] g / mL. The compounds 1 and 2 have the potential of becoming novel gram-negative bacterium prevention and treatment drugs, and are expected to provide a new direction for solving the problems in the field. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of natural drug chemistry / microbial medicine technology, and in particular to a novel polyketide compound extracted from a fermentation product of the fungus Fusarium asiaticum (F. asiaticum), a preparation method thereof, and an application thereof in resisting Gram-negative bacteria. Background Art

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

[0003] Related research on F.asiaticum has been documented, and the fungus can be isolated from wheat (Zhou Yongjin et al., Journal of Jiangsu Agricultural Sciences, 2012, 5, 979-985) and corn (A. Kawakami et al., Journal of General Plant Pathology, 2015, 81(4), 324-327). It is worth noting that there are relatively few systematic research reports on secondary metabolites of F.asiaticum. The present invention successfully isolated novel polyketide compounds with anti-Gram-negative bacterial activity from the fungus. Summary of the invention

[0004] The object of the present invention is to provide a novel polyketide compound extracted from the fermentation product of the fungus Fusarium asiaticum (F. asiaticum), a preparation method thereof and an application thereof in resisting Gram-negative bacteria.

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

[0006] A polyketone compound, the polyketone compound is represented by formula I

[0007]

[0008] Wherein, compound 1: R = -CHO, compound 2: R = -CH = CHCH3.

[0009] A method for preparing the polyketone compound:

[0010] 1) inoculating the fungus Fusarium asiaticum into a rice solid culture medium, fermenting at room temperature for 30 days, repeatedly soaking and extracting the product after the fermentation with ethyl acetate, combining the extracts and concentrating to obtain a fermentation crude extract;

[0011] 2) separating the crude extract by vacuum silica gel column chromatography, using a gradient of petroleum ether-ethyl acetate from 20:1 to 1:1 (v / v) and a gradient of dichloromethane-methanol from 20:1 to 1:1 (v / v) as solvents for gradient elution, collecting the fractions eluted by petroleum ether-ethyl acetate 1:1, and performing reverse phase silica gel (RP-18) column chromatography, eluting with methanol-water from 10:90 to 100:0;

[0012] 3) Collect the methanol-water 60:40 fraction in step 2) and conduct normal phase silica gel column chromatography, elute with dichloromethane-methanol in a ratio of 200:1 to 10:1, collect the dichloromethane-methanol 150:1 fraction, and separate and purify by preparative TLC to obtain the purified target compound 1 (molecular formula C) of formula I 22 H 30 O3);

[0013] 4) Collect the methanol-water 90:10 fractions in step 2) and purify them through gel LH-20 (methanol) to obtain the purified target compound 2 (molecular formula C 24 H 34 O2).

[0014] Specifically:

[0015] 1) The fungus F.asiaticum was streaked and inoculated on a PDA medium, cultured at 28°C for 7 days, and a 2.5 cm × 2.5 cm bacterial block was inoculated into a sterilized rice solid medium, so that the bacterial block was fully in contact with the rice medium. After the inoculation, the culture was allowed to stand at room temperature for 30 days to obtain a fermentation product, and the fermentation product was repeatedly soaked and extracted with ethyl acetate, and the extracts were combined and concentrated under reduced pressure to obtain a fermentation crude extract;

[0016] The growth characteristics of the F. asiaticum are that white fluffy aerial hyphae grow on a potato sucrose agar (PDA) medium, the initial growth is relatively slow, and then it gradually spreads to form a dense colony, and the bacterial film turns pink in the later stage. The strain has been reported in many documents and can be purchased from the Agricultural Culture Collection of China (ACCC, with a collection number of ACCC 39255) through circulation (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 gradient elution was performed using a gradient of petroleum ether-ethyl acetate from 20:1 to 1:1 (v / v) and a gradient of dichloromethane-methanol from 20:1 to 1:1 (v / v) as solvents, and the fractions eluted by petroleum ether-ethyl acetate 1:1 were collected and subjected to reverse phase silica gel column (RP-18) chromatography, and eluted with methanol-water from 10:90 to 100:0;

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

[0019] 4) Collect the eluted fractions of methanol-water 90:10 in the above step 2) and purify them by gel LH-20 (methanol) to obtain the purified target compound 2.

[0020] The rice solid culture medium has a formula of 70 grams of rice, 0.2 grams of corn steep liquor and 0.3 grams of peptone in every 100 milliliters of distilled water.

[0021] An application of the polyketide compound, and an application of the polyketide compound shown in formula I in inhibiting Gram-negative bacteria.

[0022] The polyketide compound shown in formula I is used in the preparation of drugs for controlling Gram-negative pathogenic bacteria.

[0023] The Gram-negative pathogenic bacteria are Aeromonas hydrophila, Edwardsiella ictarda, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahemolyticus or Vibrio vulnificus.

[0024] The advantages of the present invention are:

[0025] The present invention utilizes the Fusarium fungus F.asiaticum for fermentation culture, and after optimizing the culture conditions, two novel polyketide compounds are separated from the fermentation product of rice solid culture medium. Up to now, there are no reports on the chemical structure and anti-Gram-negative pathogen activity of these two compounds. Compared with normal Fusarium, the polyene side chain of compound 2 lacks a carbon atom, which may be due to the lack of S-adenosyl-L-methionine-methylation during the biosynthesis process, which is an uncommon feature that has not been reported.

[0026] The results of the Gram-negative pathogenic bacteria inhibitory activity test showed that compounds 1 and 2 had significant inhibitory activity against Gram-negative bacteria, especially compound 1 against Aeromonas hydrophila A. hydrophilia, Vibrio alginolyticus V. alginolyticus and Vibrio harveyi V. harveyi, and compound 2 against Vibrio harveyi V. harveyi, and are expected to be used in the preparation and development of a new type of drug against Gram-negative pathogenic bacteria. DETAILED DESCRIPTION

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

[0028] The present invention obtains the following compounds in the examples by isolating the Fusarium asiaticum, and the chemical structure of the compounds is as follows (the Arabic numerals in the structural formula are the positions of the carbon atoms in the chemical structure):

[0029]

[0030] Wherein, compound 1: R = -CHO, compound 2: R = -CH = CHCH3.

[0031] The growth characteristics of the Fusarium fungus F. asiaticum are white fluffy aerial hyphae growing on potato sucrose agar (PDA) culture medium. The initial growth is relatively slow, and then it will gradually spread to form dense colonies, and the mycofilm will turn pink in the later stage.

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

[0033] First, the fungus Fusarium asiaticum (2.5 cm×2.5 cm fungus block) grown on a PDA plate for 7 days was inoculated into a sterilized rice solid culture medium, so that the fungus block and the rice culture medium were fully in contact, and after the inoculation, the culture was allowed to stand at room temperature for 30 days, and the fermentation product was repeatedly soaked and extracted with ethyl acetate, and the extracts were combined and concentrated to obtain a fermentation crude extract;

[0034] The rice solid culture medium has a formula of 70 grams of rice, 0.2 grams of corn steep liquor and 0.3 grams of peptone in every 100 milliliters of distilled water.

[0035] The crude extract was subjected to reduced pressure silica gel (100-200 mesh) column chromatography (silica gel column inner diameter 10 cm), and gradient elution was performed using a gradient of petroleum ether-ethyl acetate from 20:1 to 1:1 (v / v) and a gradient of dichloromethane-methanol from 20:1 to 1:1 (v / v) as solvents in sequence;

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

[0037] The above methanol-water 90:10 components were collected and purified by gel LH-20 (methanol) to obtain the purified target compound 2; its structure was identified as shown in Formula I,

[0038]

[0039] Wherein, 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; NMR hydrogen and carbon spectra are shown in Table I; high resolution ESI mass spectrum m / z 341.2113 [MH] - , C 22 H 29 The calculated value of O3 is 341.2122.

[0042] Compound 2: colorless oil, UV(MeOH)λ max (logε)242(3.04), 275(2.75)nm; ECD(0.85mM, MeOH)λ max (Δε) 283 (-3.52) nm; NMR hydrogen and carbon spectra are shown in Table I; EI mass spectrum m / z 354 [M] + , C 24 H 34 O2 calculated value is 354. Table I H NMR spectrum (500 MHz) and C NMR spectrum (125 MHz) data 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 carbon signals was determined by DEPT method.

[0045] Example 2. Gram-negative bacteria inhibitory activity.

[0046] The minimum inhibitory concentration method was used to detect the anti-Gram-negative bacterial activity of compounds 1 and 2 shown in formula I. The following 7 strains of Gram-negative pathogens were selected: Aeromonas hydrophila, Edwardsiella ictarda, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahemolyticus, and Vibrio vulnificus for anti-aquatic disease bacteria activity test.

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

[0048] Minimum inhibitory concentration (MIC) is the lowest drug concentration that can inhibit the growth of microorganisms in vitro. In a 96-well microplate, different concentrations of drugs are added to the bacterial suspension of the test bacteria. After incubation, if the indicator bacteria in a certain well grow, it means that the drug concentration in that well cannot inhibit the growth of the bacteria. The liquid in the well is turbid and the transmittance decreases significantly. On the contrary, the liquid in the well is clear and the transmittance decreases insignificantly. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in the well is the MIC of the compound.

[0049] 2) Preparation of bacterial suspension

[0050] The above-mentioned test bacteria were inoculated into culture medium (LB medium was used for Aeromonas hydrophila, Edwardsiella ictarda, Vibrio harveyi, and Vibrio parahemolyticus; TSB medium was used for Pseudomonas aeruginosa, Vibrio alginolyticus, and Vibrio vulnificus), cultured at 28°C for 24 hours, and a proper amount of bacterial suspension was pipetted into a sterile test tube, and then the bacterial suspension was adjusted to 0.5 McFarland turbidity (equivalent to 1.5×10 8 CFU / mL), and further diluted with 0.85% NaCl solution to 5×10 5 CFU / mL.

[0051] 0.5 McFarland turbidity standard:

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

[0053] 3) Sample preparation

[0054] Take about 1 mg of the sample to be tested (i.e., the aforementioned compound 1 or compound 2), dissolve it in about 100 μL of DMSO, mix it evenly, and make the final concentration reach 2560 μL / mL. Then, take 50 μL from the sample solution and transfer it to another centrifuge tube, and add 50 μL of DMSO to obtain a sample solution with a concentration reduced by half. Using the same method, 11 groups of sample solutions with concentrations reduced by half in sequence can be obtained, and the concentration gradient is: 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 μg / mL.

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

[0056] 5) MIC determination process

[0057] 5.1) Sample addition

[0058] Using aseptic operation, add sample solutions of different concentrations after multiple dilution into a sterile 96-well plate, add sample solutions to wells 1 to 11, 5 μL per well, and do not add sample to well 12 as a growth control.

[0059] 5.2) Add bacteria

[0060] The indicator bacteria suspension equivalent to 0.5 McFarland turbidimetric was diluted 1000 times with liquid culture medium (LB culture medium for Aeromonas hydrophila, Edwardsiella ictarda, Vibrio harveyi, and Vibrio parahemolyticus; TSB culture medium for Pseudomonas aeruginosa, Vibrio alginolyticus, and Vibrio vulnificus), and 95 μL was added to the 96-well plate in sequence, so that the final concentrations of the samples in the 1st to 11th wells were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, respectively. After gently shaking and mixing, the 96-well plate was sealed and placed in a 28°C incubator for 24 hours.

[0061] 5.3) Observation of results

[0062] After the culture is completed, the absorbance value of each well at a wavelength of 600nm is measured using an ELISA instrument, and the lowest sample concentration that completely inhibits the growth of the indicator bacteria in the well is the MIC of the compound. (Note: The experiment is meaningful only when the indicator bacteria in the negative control wells grow significantly; when a single jump well occurs in the experiment, the highest drug concentration that inhibits the growth of the strain should be recorded; if multiple jump wells occur, the results should not be reported and the experiment needs to be repeated.)

[0063] Table II Antibacterial activity data of compound 1 and compound 2 (MIC, μg / mL)

[0064]

[0065] The experimental results show that compounds 1 and 2 have certain antibacterial activity against the 7 tested Gram-negative bacteria, as shown in Table II, among which compound 1 has strong inhibitory activity against Aeromonas hydrophila (A.hydrophilia), Vibrio alginolyticus (V.alginolyticus) and Vibrio harveyi (V.harveyi), with MIC values ​​of 1-2μg / mL, and compound 2 has strong inhibitory effect on Vibrio harveyi (V.harveyi) and Vibrio vulnificus (V.vulnificus), with MIC values ​​of 2-4μg / mL.

[0066] The above experimental results prove that the compounds involved in the present invention have a strong inhibitory effect on Gram-negative bacteria. They can be used to prepare new Gram-negative disease bacteria prevention and control drugs and are applied in the fields of aquaculture and so on.

Claims

1. A polyketone compound, characterized in that: The polyketone compound is shown in formula I Wherein, compound 1: R = -CHO, compound 2: R = -CH = CHCH3.

2. A method for preparing a polyketone compound according to claim 1, characterized in that: 1) inoculating the fungus Fusarium asiaticum into a rice solid culture medium, fermenting at room temperature for 30 days, repeatedly soaking and extracting the product after the fermentation with ethyl acetate, combining the extracts and concentrating to obtain a fermentation crude extract; 2) separating the crude extract by vacuum silica gel column chromatography, using a gradient of petroleum ether-ethyl acetate from 20:1 to 1:1 (v / v) and a gradient of dichloromethane-methanol from 20:1 to 1:1 (v / v) as solvents for gradient elution, collecting the fractions eluted by petroleum ether-ethyl acetate 1:1, and performing reverse phase silica gel (RP-18) column chromatography, eluting with methanol-water from 10:90 to 100:0; 3) Collect the methanol-water 60:40 fraction in step 2) and conduct normal phase silica gel column chromatography, elute with dichloromethane-methanol in a ratio of 200:1 to 10:1, collect the dichloromethane-methanol 150:1 fraction, and separate and purify by preparative TLC to obtain the purified target compound 1 (8.1 mg) of formula I with R=-CHO; 4) The components of methanol-water 90:10 in step 2) were collected and purified by gel LH-20 (methanol) to obtain the purified target compound 2 (4.1 mg) of formula I with R=-CH=CHCH3.

3. The method for preparing a polyketone compound according to claim 2, characterized in that: The rice solid culture medium has a formula of 70 grams of rice, 0.2 grams of corn steep liquor and 0.3 grams of peptone in every 100 milliliters of distilled water.

4. An application of the polyketone compound according to claim 1, characterized in that: The use of the polyketide compound shown in formula I in inhibiting Gram-negative bacteria.

5. The use of the polyketone compound according to claim 4, characterized in that: The polyketide compound shown in formula I is used in the preparation of drugs for controlling Gram-negative pathogenic bacteria.

6. The use of the polyketone compound according to claim 4 or 5, characterized in that: The Gram-negative pathogenic bacteria are Aeromonas hydrophila, Edwardsiella ictarda, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahemolyticus or Vibrio vulnificus.

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