A mixed-source terpene compound and its preparation method and application
By isolating and preparing mixed-source terpenoids from the fermentation products of Penicillium Bright Red, existing antibacterial drugs are solved to solve the problem of bacterial resistance, and the significant inhibitory activity on aquatic and human pathogenic bacteria has been achieved, and the potential of novel antibiotics is achieved.
Patent Information
- Application Number
- CN202210077733.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-01-24
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2042-01-24
AI Technical Summary
Existing antibacterial drugs are difficult to cope with bacterial resistance, and the development of new antibacterial drugs has decreased, so new natural antibiotics need to be found in microbial metabolites.
Mixed terpene compounds were isolated and prepared from the fermentation product of Penicillium chermesinum, and the compounds were purified by ethyl acetate extraction, reduced pressure silica gel column chromatography, reverse phase column chromatography and preparative thin layer chromatography.
The prepared mixed-source terpene compounds have significant inhibitory activities on the aquatic pathogen Edwardia, Vibrio eel, and the human pathogen Escherichia coli, and have the potential to be novel antibiotics or their leading compounds.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial metabolites and their application in pharmaceutical research, specifically a mixed-source terpenoid compound obtained from a fermentation product of Penicillium chermesinum, a preparation method thereof, and an application thereof in antibacterial activity. Background Art
[0002] Aquatic and human pathogens pose a serious threat to human production and livelihoods, posing significant risks to human health and economic development. To address these challenges, new antimicrobial agents must be continuously developed and applied. However, international antimicrobial research and development is currently at a low point, with the number of newly marketed antimicrobial agents decreasing year by year. Existing antimicrobial agents are struggling to address bacterial resistance. Therefore, the continued search for natural antibiotics derived from microbial metabolites is of great significance.
[0003] The fungus Penicillium chermesinum can be isolated from a variety of plants. The morphology of this strain has been reported in the literature, for example, from Kandelia candel (H. Huang et al., Journal of Natural Products, 2011, 74, 997–1002) and Heritiera littoralis (C. Darsih et al., RSC Advances, 2015, 5, 70595–70603). It has also been reported that fermentation of this strain can produce azaphilone compounds with inhibitory activity against α-glucosidase (H. Huang et al., Journal of Natural Products, 2011, 74, 997–1002). This present invention is the first to isolate mixed terpenoid compounds from this fungus, and the resulting compounds exhibit corresponding specific activities. Summary of the Invention
[0004] The present invention aims to provide a mixed-source terpenoid compound obtained from the fermentation product of Penicillium chermesinum, a preparation method thereof, and an application thereof in resisting aquatic and human pathogens.
[0005] To achieve the above object, the technical solution adopted by the present invention is:
[0006] A mixed terpenoid compound, the structure of which is compound 1 or compound 2 as shown in formula I; wherein the molecular formula of compound 1 is C 24 H 36 O4, the structural formula is 1 in formula I, and the molecular formula of compound 2 is C24 H 34 O4, the structural formula is 2 in formula I
[0007]
[0008] A method for preparing mixed-source terpenoid compounds:
[0009] 1) Fermenting Penicillium chermesinum in a solid culture medium, extracting the fermentation product 3-4 times with ethyl acetate, combining the extracts and concentrating to obtain a crude extract;
[0010] The Penicillium chermesinum is isolated from Pterocladiella tenuis, which has been reported in many literatures and is available from the China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 20723. In addition, those skilled in the art can also conveniently isolate it from Kandelia candel (H. Huang et al, Journal of Natural Products, 2011, 74, 997–1002) and Heritiera littoralis (C. Darsih et al, RSC Advances, 2015, 5, 70595–70603) according to the methods reported in the literature.
[0011] 2) subjecting the crude extract from step 1) to reduced pressure silica gel column chromatography, eluting sequentially with a gradient of petroleum ether-ethyl acetate from 50:1 to 1:1 (v / v) and a gradient of dichloromethane-methanol from 40:1 to 1:1 (v / v);
[0012] 3) The fraction eluted with a gradient of dichloromethane-methanol (40:1, v / v) in step 2) was subjected to reverse-phase column chromatography using methanol-water (10:90 to 100:0, v / v) as the eluent. The fraction eluted with methanol-water (50:50, v / v) was collected and purified by normal-phase silica gel column chromatography using a developing system of dichloromethane-methanol (100:1 to 10:1, v / v). The product was then purified by preparative thin-layer chromatography (prep. TLC) using a developing system of dichloromethane-acetone (10:1) to obtain the title compound 1.
[0013] 4) The fraction eluted with methanol-water 80:20 (v / v) in step 3) was purified by preparative thin-layer chromatography using dichloromethane-acetone (20:1, v / v) as the developing system, and then purified by gel column chromatography using methanol as the eluent to obtain the target compound 2.
[0014] Further:
[0015] 1) culturing the Penicillium chermesinum in a PDA (potato sucrose agar) medium at 28° C. for 5 days, inoculating the mycelium into a solid culture medium, and fermenting at room temperature for 30 days. The fermentation product is thoroughly soaked and repeatedly extracted with ethyl acetate, and the extracts are combined and concentrated to obtain a crude extract;
[0016] 2) The crude extract was subjected to silica gel column chromatography under reduced pressure, using a gradient elution system of petroleum ether-ethyl acetate from 50:1 to 1:1 (v / v) and dichloromethane-methanol from 40:1 to 1:1, respectively;
[0017] 3) collecting and concentrating the fractions eluted with dichloromethane-methanol 40:1 in step 2) above, and then performing reverse-phase silica gel column chromatography using a methanol-water elution system with a gradient of 10:90 to 100:0 (v / v) as the eluent;
[0018] 4) Collecting the fractions eluted with methanol and water (50:50, v / v) in step 3) above, purifying the eluted fractions by normal phase silica gel column chromatography using a developing system of dichloromethane-methanol (100:1 to 10:1, v / v), and then purifying by preparative thin layer chromatography (prep. TLC) using a developing system of dichloromethane-acetone (10:1) to obtain the mixed-source terpenoid compound 1 represented by Formula I;
[0019] 5) The fractions eluted with methanol-water (80:20, v / v) in step 3) were collected and purified by preparative thin layer chromatography (prep. TLC) using a developing system of dichloromethane-acetone (20:1, v / v), followed by purification by gel column chromatography using methanol as the eluent to obtain the mixed terpenoid compound 2 represented by Formula I.
[0020] An application of a mixed-source terpenoid compound, wherein the mixed-source terpenoid compound represented by formula I is used in the preparation of an anti-aquatic or human pathogenic bacteria preparation, or its lead compound.
[0021] The use of the mixed terpenoid compound represented by formula I in the preparation of a lead compound for resisting pathogenic bacteria in aquatic products or humans.
[0022] The aquatic pathogenic bacteria are Edwardsiella tarda and Vibrio anguillarum; the human pathogenic bacteria are Escherichia coli
[0023] The advantages of the present invention are:
[0024] 1. The mixed-source terpenoid compound prepared in the present invention is derived from the fermentation product of Penicillium chermesinum. The compound is prepared by a microbial fermentation method and is highly efficient and environmentally friendly.
[0025] 2. The mixed-source terpenoid compounds prepared by this invention exhibit significant activity against the aquatic pathogens Edwardsiella tarda and Vibrio anguillarum, as well as the human pathogen Escherichia coli. These compounds are novel and have not yet been reported, paving the way for further exploration of their mechanisms of action with the goal of developing them into novel antibiotics or lead compounds. Experiments have demonstrated that compound 1 exhibits strong inhibitory activity against the aquatic pathogens Edwardsiella tarda and Vibrio anguillarum, while compound 2 exhibits strong inhibitory activity against the human pathogen Escherichia coli. DETAILED DESCRIPTION
[0026] The present invention will be further described below with reference to some non-limiting specific implementation examples.
[0027] The present invention discovered two mixed-source terpenoid compounds from the fungus Penicillium chermesinum. The obtained compounds are novel compounds and have outstanding inhibitory activity against aquatic and human pathogens, making them potential active molecules.
[0028] Example 1. Compound Structural Formula
[0029] The structure of the mixed-source terpenoid compound isolated from the fermentation product of Penicillium chermesinum is shown in formula (I) (the Arabic numerals in the formula represent the carbon atom positions in the chemical structure):
[0030]
[0031] Example 2. Preparation of the compound represented by formula I
[0032] 1) Fermentation culture
[0033] Inoculate an appropriate amount of Penicillium chermesinum on a PDA plate and incubate at 28°C for 5 days before use. The mycelium of the fungus obtained by culturing on PDA (potato sucrose agar) medium is green and produces green spores in the later stage.
[0034] The mycelium from the PDA plate was inoculated into a sterile solid culture medium and cultured at room temperature for 30 days. The fermentation product was extracted with ethyl acetate four times, and the crude fermentation extract was obtained after combining and concentrating.
[0035] The solid culture medium comprises 70 grams of rice, 0.1 grams of corn steep liquor and 0.3 grams of peptone per 100 milliliters of seawater.
[0036] 2) Isolation and purification of crude extract
[0037] The crude extract was fractionated by silica gel (100-200 mesh) column chromatography under reduced pressure, and gradient elution was performed using petroleum ether-ethyl acetate in a gradient ratio of 50:1 to 1:1 (50:1, 20:1, 10:1, 5:1, 2:1, 1:1, v / v, the same below) and dichloromethane-methanol in a gradient ratio of 40:1 to 1:1 (40:1, 20:1, 10:1, 5:1) as elution solvents; fractions eluted with dichloromethane-methanol 40:1 were collected;
[0038] The collected fractions were subjected to reverse phase silica gel column chromatography, and gradient elution was performed with methanol-water in a ratio of 10:90 to 100:0;
[0039] The methanol-water 50:50 fraction in the above step was collected, and the eluted fraction was purified by normal phase silica gel column chromatography using a dichloromethane-methanol (100:1 to 10:1, v / v) elution system, and then purified by preparative thin layer chromatography (prep.TLC) using a dichloromethane-acetone (10:1) development system to obtain the mixed terpenoid compound 1 represented by formula I;
[0040] The methanol-water 80:20 fraction in the above step was collected and purified by preparative thin layer chromatography (prep.TLC) using a developing system of dichloromethane-acetone (20:1, v / v), and then purified by gel column chromatography using methanol as the eluent to obtain the mixed terpenoid compound 2 represented by formula I.
[0041] The compound has the following physicochemical and spectroscopic properties:
[0042] Compound 1: colorless crystal, chemical formula C 24 H 36 O4, specific rotation [α] 25 D-60(c 0.10,MeOH); UV(MeOH)λmax (logε)247(3.77),298(3.43); ECD(2.57mM,MeOH)λ max (Δε)217(-0.69),241(+0.56),291(+0.90)326(-1.80)nm; ESI-MS m / z 389.27[M+H] + ;HR-ESI-MS m / z 389.2680[M+H] + (calcd for C 24 H 37 O4,289.2514). 1 H-NMR and 13 C-NMR is shown in Table 1.
[0043] Compound 2: colorless crystal, chemical formula C 24 H 34 O4, specific rotation [α] 25 D-50(c 0.10,MeOH); UV(MeOH)λ max (logε)247(3.81),294(3.52); ECD(2.59mM,MeOH)λ max (Δε)217(-1.85),241(+1.56),289(+1.88),326(-3.87)nm; ESI-MS m / z 387.25[M+H] + ;HR-ESI-MS m / z 387.2530[M+H] + (calcd for C 24 H 35 O4,387.2457). 1 H-NMR and 13 C-NMR is shown in Table 1.
[0044] The above-mentioned compounds can also be obtained by organic synthesis.
[0045] Table 1. H NMR (500 MHz, DMSO-d6) and C NMR (125 MHz, DMSO-d6) data
[0046]
[0047] Example 3. Inhibitory activity against aquatic and human pathogens
[0048] The minimum inhibitory concentration method was used to test the antibacterial activity of compounds 1 and 2 of Formula I against aquatic and human pathogens. Two strains of aquatic pathogens, Edwardsiella tarda and Vibrio anguillarum, and one strain of human pathogen Escherichia coli were selected for antibacterial activity testing.
[0049] 1) Antibacterial activity test (MIC method):
[0050] The minimum inhibitory concentration (MIC) is the lowest drug concentration that inhibits bacterial growth in vitro. In a 96-well microplate, varying concentrations of drug are added to a suspension of the test bacteria. After incubation and observation, if the indicator bacteria grow in a particular well, the drug concentration in that well is insufficient to inhibit its growth. The liquid in that well becomes turbid, with a significant decrease in light transmittance. Conversely, the liquid in that well becomes clear, with no significant decrease in light transmittance. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in a well is the compound's MIC.
[0051] 2) Preparation of bacterial suspension
[0052] The above test strains were inoculated into culture medium (TSB medium for Edwardsiella tarda, LB medium for Vibrio anguillarum and Escherichia coli) and cultured in a shaking incubator at 37°C for 12 hours. An appropriate amount of bacterial suspension was pipetted into a sterile test tube and diluted with 0.85% NaCl solution to a McFarland turbidity of 0.5 (equivalent to 1.5×10 8 CFU / mL), 0.5 McFarland turbidity standard:
[0053] 0.5 mL x 0.048 mol / L BaCl2 (1.175% w / v BaCl2·2H2O) was added to 99.5 mL x 0.18 mol / L (0.36 N) H2SO4 (1% v / v) and stirred continuously to maintain a suspension.
[0054] 3) Sample preparation
[0055] Dissolve approximately 1 mg of each test sample (Compound 1 and Compound 2 obtained above) and the positive control (chloramphenicol) in approximately 1 mL of DMSO. Mix thoroughly to a final concentration of 1280 μg / mL. Pipette 500 μL of the sample solution into another centrifuge tube, followed by the addition of 500 μL of DMSO to obtain a sample solution with a concentration reduced by half. This method yielded a total of nine sets of sample solutions with concentrations reduced by half (1280, 640, 320, 160, 80, 40, 20, 10, and 5 μg / mL).
[0056] 4) Blank control: Select pure solvent that does not dissolve the sample to be tested as the blank control.
[0057] 5) MIC determination process
[0058] 5.1) Aseptically add serially diluted sample solutions of varying concentrations to a sterile 96-well plate. Add 5 μL of sample solution to wells 1 through 9, leaving well 10 empty as a growth control. Each sample should be plated in triplicate.
[0059] 5.2) Dilute the indicator bacteria suspension to a McFarland turbidity of 0.5 1000-fold with liquid culture medium (TSB medium for Edwardsiella tarda, LB medium for Vibrio anguillarum and Escherichia coli). Add 95 μL of the suspension to a 96-well plate, so that the final concentrations in wells 1 to 9 are 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively. Gently shake to mix, seal the 96-well plate, and place it in a 37°C incubator. Incubate for 12 hours before observation.
[0060] 5.3) Measure the absorbance of each well at 600 nm using a microplate reader. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in the well is the compound's MIC. (Note: The experiment is only meaningful when the indicator bacteria clearly grow in the growth control wells. If a single well skips, the highest drug concentration that inhibits growth should be recorded. If multiple wells skip, the result should not be reported and the experiment should be repeated.)
[0061] The experimental results showed that compound 1 exhibited significant inhibitory activity against the aquatic pathogens Edwardsiella tarda and Vibrio anguillarum, with minimum inhibitory concentrations (MICs) of 0.5 μg / mL, compared to the positive control chloramphenicol, which had MICs of 0.5 and 1 μg / mL, respectively. Compound 2 also exhibited significant inhibitory activity against the human pathogen Escherichia coli, with a minimum inhibitory concentration (MIC) of 1.0 μg / mL, compared to the positive control chloramphenicol, which had a MIC of 2 μg / mL.
[0062] The above experimental results demonstrate that the compounds of the present invention have a strong inhibitory effect on aquatic and human pathogenic bacteria, and they can be used to prepare new antibiotics or their lead compounds.
Claims
1. A mixed-source terpene compound, characterized in that: The structure of the mixed terpene compound is as shown in compound 2, and the molecular formula of compound 2 is C 24 H 34 O4, the structural formula is as follows: 。 2. A method for preparing a mixed-source terpene compound, characterized in that: The structural formula of the mixed-source terpene compound is shown in Formula I: 1) Penicillium rubrum Penicillium chermesinum Fermenting the product in a solid medium, then extracting the fermentation product with ethyl acetate for 3-4 times, combining the extracts and concentrating them to obtain a crude extract; 2) subjecting the crude extract in step 1) to reduced pressure silica gel column chromatography, eluting with a gradient of petroleum ether-ethyl acetate from v / v 50:1 to 1:1 and a gradient of dichloromethane-methanol from v / v 40:1 to 1:1; 3) The fraction eluted with a gradient of dichloromethane-methanol v / v 40:1 in step 2) was subjected to reverse phase column chromatography using methanol-water v / v 10:90 to 100:0 as the eluent, and the fraction eluted with methanol-water v / v 50:50 was collected and purified by normal phase silica gel column chromatography using a developing system of dichloromethane-methanol v / v 100:1 to 10:1, and then purified by preparative thin layer chromatography using a developing system of dichloromethane-acetone 10:1 to obtain the target compound 1; 4) The component eluted with methanol-water v / v 80:20 in step 3) was purified by preparative thin layer chromatography using a developing system of dichloromethane-acetone v / v 20:1, and then purified by gel column chromatography using methanol as the eluent to obtain the target compound 2.
3. The method for preparing mixed-source terpenoid compounds according to claim 2, characterized in that: 1) the Penicillium rubrum Penicillium chermesinum The culture was carried out in a potato sucrose agar PDA medium at 28°C for 5 days, the mycelium was inoculated into a solid medium, and the culture was allowed to ferment at room temperature for 30 days, the fermentation product was fully soaked and repeatedly extracted with ethyl acetate, and the extracts were combined and concentrated to obtain a crude extract; 2) subjecting the crude extract to reduced pressure silica gel column chromatography, using a gradient elution system of petroleum ether-ethyl acetate in a range of v / v 50:1 to 1:1 and a gradient elution system of dichloromethane-methanol in a range of v / v 40:1 to 1:1, respectively; 3) collecting and concentrating the fractions eluted with dichloromethane-methanol 40:1 in the above step 2), and performing reverse phase silica gel column chromatography after concentration using a methanol-water elution system with a gradient of v / v 10:90 to 100:0 as an eluent; 4) collecting the fractions eluted with methanol-water v / v 50:50 in the above step 3), purifying the eluted fractions by normal phase silica gel column chromatography, using an elution system of dichloromethane-methanol v / v 100:1 to 10:1, and then purifying by preparative thin layer chromatography, using a developing system of dichloromethane-acetone 10:1, to obtain a mixed terpene compound 1 represented by formula I; 5) Collect the components eluted with methanol-water v / v 80:20 in the above step 3), purify them by preparative thin layer chromatography using a developing system of dichloromethane-acetone v / v 20:1, and then purify them by gel column chromatography using methanol as the eluent to obtain the mixed terpene compound 2 represented by formula I.
4. An application of the mixed-source terpene compound according to claim 1, characterized in that: The compound 2 is used in the preparation of anti-human pathogenic bacteria Escherichia coli Escherichia coli Application in preparations.