Process for preparing a derivative of asperbenin and use thereof

Abstract

The application provides a fusariobutenolide derivative 4''-O-isopentenyl fusariobutenolide C, and a structural formula of the compound is provided.The 4''-O-isopentenyl fusariobutenolide C can significantly inhibit the growth of seven human routine pathogenic bacteria in antibacterial activity testing, and the MIC is 8.6-28.3 μg / mL, and the antibacterial effect on one strain of human drug-resistant pathogenic bacteria, methicillin-resistant Staphylococcus aureus, is significant, and the MIC is 16.8 μg / mL, and the 4''-O-isopentenyl fusariobutenolide C can be applied to preparation of antibacterial drug precursors, and lays a material foundation for next-step biological medicine development and application of antibacterial components.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural medicine, and in particular relates to a preparation method of asperbenzolide derivative and antibacterial application thereof. BACKGROUND

[0002] The discovery and application of antibiotics is a distinguished contribution in the history of human medicine. Since the discovery of penicillin in nature, bacterial infections have been effectively controlled. However, with the wide application of antibiotics in the world, sensitive strains are gradually eliminated under the selective pressure of antibiotics, and drug-resistant strains become dominant, and drug resistance can be transmitted between different species at the genetic level, making the infection very difficult, and the available antibiotics in the clinic are increasingly scarce, making the situation of drug-resistant bacterial infection more severe. The treatment effect of traditional antibiotics such as penicillin on infections caused by drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has been greatly reduced, resulting in an increase in clinical complications and deaths.

[0003] The research and development of new antibacterial drugs show considerable potential for the treatment of drug-resistant bacterial infections. The structure and target of new antibacterial drugs are different from traditional antibiotics, so they can have good efficacy on drug-resistant bacteria. Therefore, the World Health Organization (WHO) has developed new antibacterial drugs as a major strategy to combat drug-resistant bacterial infections. Under the policy incentive of new drug research and development, the research and development of new antibacterial drugs have been valued by new drug research and development institutions. Fungus-derived antibiotics such as cephalosporin have significant antibacterial effects and low toxicity to the human body. Therefore, accelerating the research of new antibacterial drugs, discovering new structure of fungus-derived antibacterial substances, and studying their antibacterial effects on drug-resistant bacteria to provide clinically structural and effective drug compounds are problems that need to be solved by those skilled in the art. SUMMARY

[0004] The present application is based on at least one of the above technical problems, and proposes a novel structure asperbenzolide derivative 4''-O-isopentenyl asperbenzolide C with significant antibacterial effect and a preparation method thereof. The compound provides an effective drug precursor for the development of antibacterial drugs and lays a material foundation for further pharmacological research.

[0005] Therefore, the present application proposes a novel structure asperbenzolide derivative 4''-O-isopentenyl asperbenzolide C, which is a 4''-O-isopentenyl substituted biphenyl asperbenzolide derivative. The structural formula of the compound is

[0006] .

[0007] According to the second aspect of the present application, the application of asperbenzolide derivative 4''-O-isopentenyl asperbenzolide C in the preparation of antibacterial drugs is proposed.

[0008] According to the third aspect of the present application, a method for preparing the asperbeninolide derivative 4''-O-isopentenyl asperbeninolide C is provided, comprising the following steps:

[0009] (1) Activating the Aspergillus flavipes to form a seed liquid, and culturing and fermenting in a solid culture medium;

[0010] (2) After cold soaking extraction of the fermentation obtained in step (1), the crude extract is concentrated under reduced pressure to obtain a crude extract;

[0011] (3) The crude extract in step (2) is impurity-removed by silica gel column chromatography, then eluted by macroporous resin column chromatography, and then the target sub-component is separated and purified by high performance liquid chromatography.

[0012] Further, the preparation method of the solid culture medium in step (1) is as follows: 60-140 g of rice and 80-150 mL of water are soaked overnight, and then autoclaved at 100-120°C for 10-50 min.

[0013] Further, the elution gradient of the macroporous resin column chromatography in step (3) is 10%-100% methanol-water.

[0014] Further, the target sub-component in step (3) is the 50%-90% methanol-water section.

[0015] Further, the high performance liquid chromatography eluent is 45%-90% methanol-acid water.

[0016] The present application provides a kind of asperbeninolide derivative 4''-O-isopentenyl asperbeninolide C and its antibacterial effect, with the following technical effects: the present application provides a kind of novel structure type asperbeninolide derivative, subsequent research finds that 4''-O-isopentenyl asperbeninolide C can significantly inhibit the growth of 7 strains of human routine pathogenic bacteria, and the MIC is 8.6-28.3 μg / mL, and the antibacterial effect on 1 strain of human drug-resistant pathogenic bacteria methicillin-resistant Staphylococcus aureus is significant, and the MIC is 16.8 μg / mL.4''-O-isopentenyl asperbeninolide C can be applied in the preparation of antibacterial drugs, and lays a material foundation for the next step of pharmacodynamic research and drug development. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The colony morphology of Aspergillus flavipes is shown

[0018] Figure 2 The key HMBC signals of 4''-O-isopentenyl asperbeninolide C are shown.

[0019] Figure 3 The 1H NMR spectrum of 4''-O-isopentenyl asperbeninolide C is shown.1 H-NMR spectrum.

[0020] Figure 4 The following is an illustration of 4''-O-isopentenyl aspergillin C. 13 C-NMR spectrum.

[0021] Figure 5 The diagram shows the antibacterial effect of 4''-O-isopentenyl aspergillin C on Escherichia coli and Pseudomonas aeruginosa.

[0022] Figure 6 The diagram shows the antibacterial effect of 4''-O-isopentenyl aspergillin C against clinical strains of Staphylococcus aureus and Klebsiella pneumoniae.

[0023] Figure 7 The antibacterial effect of 4''-O-isopentenyl aspergillin C on MRSA is shown in the figure. Detailed Implementation

[0024] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0025] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0026] Example 1: Fermentation and Extraction of Microbial Strains

[0027] Aspergillus flavus ( Aspergillus flavipes The strain was purchased from the American Type Culture Collection (ATCC), accession number ATCC 200802. The strain was revived by streak plating and inoculated onto PDA solid medium, where it was activated for 5 days at 28°C. Small colony pieces were surgically removed from the PDA medium and inoculated onto PDB liquid medium, then cultured on a shaker at 28°C and 130 rpm for 5 days to obtain a seed culture. This seed culture was then inoculated onto rice solid medium and cultured statically at 28°C and humidity for 30 days. Rice fermentation product was extracted twice with 400 mL of ethyl acetate per 200 g of fermentation product using cold extraction. The extracts were combined and concentrated under reduced pressure until no ethyl acetate odor remained, yielding an ethyl acetate extract.

[0028] Example 2: Isolation and identification of 4''-O-isopentenyl aspergillin C

[0029] The extract was fractionated on a silica gel column and eluted with petroleum ether-ethyl acetate (40:1 to 1:3) in sections (A-I). Fractions E-H were combined and applied to a MCI column and eluted with 10%, 30%, 50%, 70%, 90%, 100% methanol-water gradient. Each fraction was combined and evaporated to dryness. The fourth fraction (70% methanol-water fraction) was purified by semi-preparative HPLC with the following conditions: mobile phase: 60% methanol-water + 0.1% CF3COOH, flow rate: 4.0 mL / min, detection wavelength: 210 nm, peak elution time: 52.5-54.8 min. The peak was collected and evaporated to dryness to give a white solid (12.5 mg). The white solid was analyzed by mass spectrometry and one- and two-dimensional nuclear magnetic resonance to determine the structure of the compound as Low resolution mass spectrometry in positive ion mode [M+Na] + m / z 529.3, [M+NH4] + m / z 524.5, from which the molecular weight of the compound was determined to be 506, and the molecular formula of the compound was determined to be C 30 H 34 O 7。 Its 1 H and 13 C nuclear magnetic resonance data are shown in Table 1. From 1 H and 13 C nuclear magnetic resonance data, the compound was determined to have two carbonyl groups (C-1 and C-6), 18 olefinic carbons (C-2, C-3, C-1', C-2', C-3', C-4', C-5', C-6', and C-1'', C-2'', C-3'', C-4'', C-5'', C-6'', C-8'', C-9'', C-13'', C-14''), one quaternary carbon with an oxygen (C-4), three methylenes (C-5, C-7'', C-12''), two methoxyl groups (OCH3-2, OCH3-6), and four methyl groups (C-10'', C-11'', C-15'', C-16''). The signals were assigned by two-dimensional nuclear magnetic resonance data such as HSQC and HMBC. The butenolide ring was confirmed by HMBC signals from H-5 to C-1 / C-3 / C-4 / C-6, and the para-substituted benzene ring was confirmed by H-2' and H-6' (7.54, 2H, d, J = 8.7 Hz), H-3' and H-5' (6.90, 2H, d, J= 8.7 Hz), and HMBC signals from H-2' to C-3 / C-1' / C-3' / C-4' and H-5' to C-1' / C-3' / C-4' confirmed the position of the meta-trisubstituted benzene ring by its hydrogen coupling and HMBC signals from H-5 to C-2'' / C-6'', H-2'' to C-5 / C-1'' / C-3'' / C-4'' / C-6'', and H-5'' to C-1'' / C-3'' / C-4'' / C-6''. The 3''-isopentenyl group was identified by H-7'' to C-2'' / C-3'' / C-4'' and H-2'' to C-7'', and the 4''-O-isopentenyl group was identified by H-12'' to C-4''. The structure of the compound was thus determined. The 4''-O-isopentenyl aspergillin lactone C proposed in this invention is a novel compound in which the 4''-phenolic hydroxyl group of aspergillin lactone C is replaced by an isopentenyl group.

[0030] Table 1. Compounds 1 H (500MHz) and 13 C10 (125MHz) NMR data (CDCl3)

[0031]

[0032] Example 3: Inhibition experiment on three common strains of Gram-positive human pathogens

[0033] The human pathogenic bacteria used in the antibacterial experiment were fecal cocci ( Enterococcus faecalis ATCC 29212), Bacillus subtilis ( Bacillus subtilis ATCC 6633), Bacillus hygroscopicus ( B. altitudinis The bacteria were activated on beef extract peptone agar plates at 37°C for 24 h. Colonies were picked and added to MH broth (Mueller-Hinton-Broth), and cultured with shaking for 6 h. The bacterial culture was then diluted to 1.0 × 10⁻⁶. 4 ~1.0 × 10 5CFU / mL was prepared for use. 4''-O-isopentenyl aspergillin C was dissolved in DMSO and diluted with MH culture medium to prepare test solutions of 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL. For the blank group, 200 μL of MH culture medium was added; for the positive group, 100 μL of positive drug solution and 100 μL of bacterial suspension were added; for the test group, 100 μL of test sample solution and 100 μL of bacterial suspension were added; and for the growth group, 100 μL of MH culture medium and 100 μL of bacterial suspension were added. After incubation at 37°C for 24 h, the OD value at 530 nm was measured using a microplate reader, with three replicates for each group. The MIC (minimum inhibitory concentration) was the drug concentration at which the bacterial OD value decreased by half. Statistical analysis was performed using GraphPad Prism 8 software to calculate the MIC value and ±SD (standard deviation). The results showed that 4''-O-isopentenyl aspergillin C had a significant inhibitory effect on three human pathogenic bacteria, with a MIC of 13.0–17.3 μg / mL.

[0034] Table 2. MIC (μg / mL) of 4''-O-isopentenyl aspergillin C against three human pathogenic bacteria.

[0035]

[0036] Example 4: Antibacterial effect against two common Gram-negative human pathogenic strains

[0037] The bacterial strain used is Escherichia coli (E. coli) E. coli ATCC 25922) and Pseudomonas aeruginosa ( P. aeruginosa ATCC27853 was activated on beef extract peptone agar plates at 37°C for 24 h. Colonies were picked and added to MH broth, shaken and incubated for 6 h. The bacterial suspension was then diluted for later use. 4''-O-isopentenyl aspergillus lactone C was dissolved in DMSO and diluted with MH broth to prepare test solutions of 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL. 100 μL of different concentrations of compound solution and 100 μL of bacterial suspension were added to the test group. After incubation at 37°C for 24 h, the OD value at 530 nm was measured using a microplate reader. Each group was tested in triplicate. The results showed that ( Figure 5The inhibition rate of 12.5 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against *Escherichia coli* reached 69.5%, and the inhibition rate of 6.25 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against *Escherichia coli* was 30.2%. Therefore, the MIC of 4''-O-isopentenyl aspergillus butyrolactone C against *Escherichia coli* was calculated to be 8.7 μg / mL. The inhibition rate of 12.5 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against *Pseudomonas aeruginosa* reached 56.0%, and the inhibition rate of 6.25 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against *Pseudomonas aeruginosa* was 30.1%. Therefore, the MIC of 4''-O-isopentenyl aspergillus butyrolactone C against *Pseudomonas aeruginosa* was calculated to be 10.7 μg / mL. 4''-O-isopentenyl aspergillin C showed significant antibacterial effects against two strains of common human pathogens that are Gram-negative.

[0038] Example 5: Antibacterial effect against two clinically pathogenic human strains

[0039] clinical strains of Staphylococcus aureus ( S. aureus clinical isolate) and Klebsiella pneumoniae clinical strain ( K. pneumonia Clinical isolate (Clinical isolate) was activated at 37°C for 24 h on beef extract peptone agar plates. Colonies were picked and added to MH culture medium, and cultured with shaking for 6 h. The bacterial suspension was then diluted for later use. 4''-O-isopentenyl aspergillus lactone C was dissolved in DMSO and diluted with MH culture medium to prepare test solutions of 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL. 100 μL of different concentrations of compound solution and 100 μL of bacterial suspension were added to the test group. After culturing at 37°C for 24 h, the OD value at 530 nm was measured using a microplate reader. Each group was tested in triplicate. The results showed that ( Figure 6The inhibition rate of 12.5 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Staphylococcus aureus reached 68.0%, and the inhibition rate of 6.25 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Staphylococcus aureus reached 35.9%. Therefore, the MIC of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Staphylococcus aureus was calculated to be 8.6 μg / mL. The inhibition rate of 50.0 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Klebsiella pneumoniae reached 63.6%, and the inhibition rate of 25.0 μg / mL of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Klebsiella pneumoniae was 36.7%. Therefore, the MIC of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Klebsiella pneumoniae was calculated to be 28.3 μg / mL. 4''-O-isopentenyl aspergillin C showed significant antibacterial effects against two clinical strains of common human pathogens.

[0040] Example 6: Antibacterial effect against human pathogenic drug-resistant MRSA strains

[0041] Methicillin-resistant Staphylococcus aureus Staphylococcus aureus MRSA was activated on beef extract peptone agar plates at 37°C for 24 h. Colonies were picked and added to MH broth, shaken and incubated for 6 h. The bacterial suspension was then diluted for later use. 4''-O-isopentenyl aspergillus lactone C was dissolved in DMSO and diluted with MH broth to prepare test solutions of 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL, using tigecycline as the positive control. 100 μL of different concentrations of compound solution and 100 μL of bacterial suspension were added to the test group. After incubation at 37°C for 24 h, the OD value at 530 nm was measured using a microplate reader. Each group was tested in triplicate. The results showed that ( Figure 7 The inhibition rate of 4''-O-isopentenyl aspergillus butyrolactone C at 25.0 μg / mL against MRSA reached 76.0%, and the inhibition rate of 4''-O-isopentenyl aspergillus butyrolactone C at 12.5 μg / mL against MRSA was 27.5%. Therefore, the MIC of 4''-O-isopentenyl aspergillus butyrolactone C against clinical strains of Staphylococcus aureus was calculated to be 16.8 μg / mL. 4''-O-isopentenyl aspergillus butyrolactone C showed significant antibacterial effect against human pathogenic drug-resistant MRSA strains.

[0042] In summary, the present application proposes a new structure of asperbenzolide C phenolic hydroxyl group is substituted by isopentenyl compound 4''-O-isopentenyl asperbenzolide C, subsequent research found that 4''-O-isopentenyl asperbenzolide C can significantly inhibit the growth of 7 strains of human pathogenic bacteria, MIC is 8.6-28.3 μg / mL, and the antibacterial effect of 1 strain of human drug-resistant pathogenic bacteria methicillin-resistant Staphylococcus aureus is significant, MIC is 16.8 μg / mL, 4''-O-isopentenyl asperbenzolide C can be applied to the preparation of antibacterial drugs, and lays a material foundation for the next biological medicine development and application of antibacterial components.

[0043] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An asperbenzinolide derivative 4"-O-isopentenyl asperbenzinolide C, characterized by, The structural formula of the compound is 。 2. Use of asperbenin derivative 4"-O-isopentenyl asperbenin C according to claim 1 in the preparation of an antibacterial medicament.

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