Fungus citranaxol a, preparation method and application thereof

Abstract

The application provides a fungal tangeretin derivative tangeretin A, and a structural formula of the compound is The tangeretin A provided by the application is a tangeretin derivative with a novel skeleton structure, is a tetraloop novel skeleton type, has a rare spiro skeleton, is a polymer formed by a quinone tangeretin and 2-hexene 1,3-diene-2,5-dimethyl furanone in terms of biogenesis, and can be applied to preparation of human pathogenic bacteria and an agricultural antibiotic precursor, and lays a material foundation for next-step biological medicine and pesticide research and application of an antibacterial component.

Description

Technical Field

[0001] This application belongs to the field of natural medicine technology, and specifically relates to a fungus, citrione A, its preparation method and application. Background Technology

[0002] The discovery and application of antibiotics is a remarkable contribution to the history of human medicine. Since the discovery of penicillin in nature, bacterial infections have been effectively controlled. However, with the widespread use of antibiotics worldwide, sensitive bacterial strains have gradually been eliminated under the selective pressure of antibiotics, while drug-resistant strains have become the dominant bacterial group. Furthermore, drug resistance can spread between different species at the genetic level, making infections extremely difficult to manage. This has led to a growing shortage of clinically available antibiotics, resulting in an increasingly severe situation regarding drug-resistant bacterial infections. The therapeutic efficacy of traditional antibiotics such as penicillin against infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant bacteria has been greatly reduced, leading to an increase in clinical complications and deaths.

[0003] The development of novel antimicrobial drugs shows considerable potential for treating drug-resistant bacterial infections. These novel drugs differ in structure and target mechanism from traditional antibiotics, thus exhibiting better efficacy against drug-resistant bacteria. Currently, fungal antibiotics, represented by cephalosporins, are experiencing rapid development. Fungal antibiotics have significant antibacterial effects and low toxicity to humans. Recent studies have shown that fungi can produce antimicrobial active ingredients such as alkaloids, terpenes, and polyketides, and their numbers are gradually increasing, making them a hot topic in the discovery and development of antimicrobial active ingredients. Therefore, accelerating the research of new antimicrobial drugs, discovering novel fungal-derived antimicrobial substances, studying their antimicrobial effects against drug-resistant bacteria, and providing clinically diverse and highly effective prodrug compounds are urgent problems that need to be solved by those skilled in the art. Summary of the Invention

[0004] Based on at least one of the aforementioned technical problems, this application proposes a novel citrinin derivative with significant antibacterial activity, citrinin A, and its preparation method. This compound provides an effective drug precursor for the development of antibacterial drugs and lays a material foundation for further pharmacodynamic studies.

[0005] In view of this, this application proposes a fungal citrinin derivative, citrinin A, wherein the structural formula of citrinin A is:

[0006]

[0007] The citrinin A is a novel citrinin derivative with a novel tetracyclic skeleton and a rare spirocyclic skeleton. Biotically, it is a polymer formed by quinone citrinin and 2-hexane-1,3-diene-2,5-dimethylfuranone.

[0008] According to a second aspect of this application, the use of the fungal citrinin derivative citrione A in the preparation of drugs against human pathogenic bacteria is proposed.

[0009] In some embodiments, the human pathogenic bacteria include standard strains of Staphylococcus aureus, clinical strains of Staphylococcus aureus, Bacillus subtilis, and fecal cocci, and the MIC of citrinum A is 22.5-67.3 μg / mL.

[0010] In some embodiments, the fungal citrinin derivative citrione A is used in the preparation of precursors for agricultural antibiotics.

[0011] In some embodiments, the agricultural antibiotic prepared from the citrione A is used to combat soft rot fungus of konjac and bacterial canker of kiwifruit.

[0012] According to a third aspect of this application, a method for preparing the citrinin derivative citrione A is provided, comprising the following steps:

[0013] (1) After activating Aspergillus cristatus, seed liquid was formed and cultured and fermented in a solid culture medium;

[0014] (2) After cold extraction of the fermentation product obtained in step (1), the crude extract was concentrated under reduced pressure.

[0015] (3) After removing impurities from the crude extract in step (2) by silica gel column chromatography, the target sub-components are eluted by macroporous resin column chromatography and then separated and purified by high performance liquid chromatography.

[0016] In some embodiments, the solid culture medium in step (1) is prepared by soaking 70-130g of rice, 80-190mL of water, and 0.02-1.0g of K2HPO4 overnight at 100-120℃ for 10-50min.

[0017] In some embodiments, the macroporous resin column chromatography elution gradient in step (3) is 10% to 100% ethanol-water.

[0018] In some embodiments, the target sub-component in step (3) is a 50% to 90% ethanol-water segment.

[0019] In some embodiments, the high performance liquid chromatography eluent is acetonitrile-water with a volume ratio of 40% to 80% acetonitrile and water.

[0020] This application proposes a novel tetracyclic skeleton type and a rare spirocyclic structure of citrinin derivative citrione A, which can be used in the preparation of drugs against human pathogenic bacteria and in the preparation of precursors for agricultural antibiotics, laying a material foundation for further pharmacodynamic studies. Attached Figure Description

[0021] Figure 1The key to citrinin A was shown. 1 H- 1 H COSY and HMBC signals;

[0022] Figure 2 The high-resolution mass spectrum of citrione A is shown;

[0023] Figure 3 The following is an example of citrione A. 1 H-NMR spectrum;

[0024] Figure 4 The following is an example of citrione A. 13 C-NMR spectrum;

[0025] Figure 5 The following is an example of citrione A. 1 H- 1 H COSY diagram;

[0026] Figure 6 The HSQC diagram of citrione A is shown;

[0027] Figure 7 The HMBC diagram of citrinin A is shown;

[0028] Figure 8 The biosynthetic derivation diagram of citrione A is shown;

[0029] Figure 9 The antibacterial effect of citrinin A on konjac soft rot fungus is shown in the figure;

[0030] Figure 10 The diagram shows the antibacterial effect of citrinin A on soft rot fungus in kiwifruit. Detailed Implementation

[0031] To better understand the above-mentioned objectives, features, and advantages of this application, the application 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 and features described in these embodiments can be combined with each other.

[0032] Many specific details are set forth in the following description in order to provide a full understanding of this application. However, this application may also be implemented in other ways different from those described herein. Therefore, the scope of protection of this application is not limited to the specific embodiments disclosed below.

[0033] Example 1: Fermentation and Extraction of Microbial Strains

[0034] *Aspergillus cristatus*, purchased from the American Type Culture Collection (ATCC), accession number ATCC 22166. The strain was revived by streak plating and inoculated onto PDA solid medium, activated for 6 days at 28°C. Small colony fragments were surgically removed from the PDA medium and inoculated onto PDB liquid medium, 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.

[0035] Example 2: Isolation and Identification of Citrinal A

[0036] The extract was fractionated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate (30:1 to 1:3). The fractions were combined to obtain 8 segments (AH). The CE segments were combined and loaded onto an MCI column, adsorbed overnight, and eluted with a gradient of 10%, 30%, 50%, 70%, 90%, and 100% ethanol-water. Each gradient was combined and evaporated to dryness to obtain 6 subfractions. The 4th subfraction (70% ethanol-water fraction) was separated and purified by semi-preparative high-performance liquid chromatography (HPLC). The mobile phase was 70% CH3CN-H2O, the flow rate was 4.0 mL / min, the detection wavelength was 210 nm, and the elution time was 41.5–43.8 min. This peak was intercepted and evaporated to dryness to obtain 9.5 mg of a white solid. Mass spectrometry and one-dimensional / two-dimensional NMR analyses of this white solid were performed as follows: Figures 2-7 As shown, where

[0037] The structure of the compound was determined to be as follows: High-resolution mass spectrometry in positive ion mode [M+Na] + m / z 421.1988, calculated value 421.1985 (C 24 H 30 From the given information (O5Na), we can determine that the molecular weight of the compound is 398, and therefore the molecular formula is C. 24 H 30 O5. Its 1 H and 13 The C NMR data are shown in Table 1. (From...) 1 H and 13C10 NMR data revealed that the compound contains one carbonyl group (C-3'), ten alkene carbons (C-4a, C-5, C-6, C-7, C-8, C-8a and C-8', C-9', C-10', C-11'), one hemiketal carbon (C-5'), one oxygen-linked quaternary carbon (C-2'), three methine carbons (C-1, C-3, C-4), three methylene groups (C-4', C-6', C-12'), and five methyl groups (C-9, C-10, C-11, C-7', C-13'). 1 H- 1 The signal was assigned using two-dimensional NMR data from H COSY, HSQC, and HMBC. The A and B rings of the azophenone fragment are derived from H3-9 / H-3 / H-4 / H3-10. 1 H- 1 The H COSY signal and HMBC signals from H-1 to C-3 / C-4a / C-8a, H-3 to C-1 / C-4 / C-4a, H-4 to C-3 / C-5 / C-8a, H-7 to C-5 / C-6 / C-8 / C-8a, H3-9 to C-3 / C-4, H3-10 to C-3 / C-4 / C-4a, and H3-11 to C-4a / C-5 / C-6 confirm the presence of furanone D rings. The furanone D ring is confirmed by H-4′ to C-2′ / C-3′ / C-5′ and H3-7′ to C-2′ / C-3′. The E,E-1,3-hexadiene side chains are... 1 H- 1 The substitution position C-2′ was confirmed by the H COSY signal, and the substitution was determined by the key HMBC signals H3-7′ to C-8′ and H3-9′ to C-2′. The C ring of the pyranone was... 1 H- 1 The presence of the spirocyclic ring is confirmed by HCOSY signal H-1 / H-6′ and HMBC signals H-1 to C-8 / C-8a / C-6′ and H-6′ to C-1 / C-8a / C-5′. The presence of the spirocyclic ring is confirmed by the hemiketal carbon and HMBC signals H-6′ to C-4 and H-4′ to C-6′. The citrione A proposed in this application is a novel tetracyclic skeleton with a rare spirocyclic skeleton, biologously a polymer formed from quinone-type citrione and 2-hexane-1,3-diene-2,5-dimethylfuranone. Figure 8 As shown.

[0038] Table 1 Compounds 1 H (500MHz) and 13 C10 (125MHz) NMR data (CDCl3)

[0039]

[0040] Example 3: Inhibition Experiment on Human Pathogenic Bacteria

[0041] The human pathogenic bacteria used in the antibacterial experiment were the standard strain of Staphylococcus aureus (ATCC25923), the clinical strain of Staphylococcus aureus (S. aureus clinical isolate), Bacillus subtilis (ATCC 6633), and Enterococcus faecalis (ATCC 29212). The bacteria were activated on beef extract peptone agar plates at 37°C for 24 hours. Colonies were picked and added to MH broth (Mueller-Hinton Broth), and cultured with shaking for 6 hours. The bacterial suspension was then diluted to 1.0 × 10⁻⁶. 4 ~1.0×10 5 CFU / mL was prepared for use. Citrus amycin A 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℃ 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 citrus amycin A had a significant inhibitory effect on four human pathogenic bacteria, with MICs ranging from 22.5 to 67.3 μg / mL.

[0042] Table 2. MIC (μg / mL) of citrinin A against four human pathogenic bacteria.

[0043]

[0044] Example 4: Antibacterial effect against agricultural pathogenic bacterium *Amorphophallus konjac*

[0045] The microbial strain *Erwinia carotovora* var. *carotovora* 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 to 1.0 × 10⁻⁶. 4 ~1.0×10 5CFU / mL was prepared for use. Citrus amycin A was dissolved in DMSO and diluted with MH 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 medium was added; for the positive group, 100 μL of positive control solution and 100 μL of bacterial suspension were added; for the test group, 100 μL of different concentrations of citrus amycin A solution and 100 μL of bacterial suspension were added; and for the growth group, 100 μL of MH medium and 100 μL of bacterial suspension were added. After incubation at 37℃ for 24 h, the OD value at 530 nm was measured using a microplate reader. Each group was tested in triplicate. Results are as follows: Figure 9 As shown, 12.5 μg / mL of citrinum A inhibited the growth of *Amorphophallus konjac* by 68.3%, and 6.25 μg / mL of citrinum A inhibited the growth of *Amorphophallus konjac* by 26.8%. Therefore, the MIC of citrinum A against *Amorphophallus konjac* was calculated to be 9.1 μg / mL. Thus, it can be concluded that the citrinum A obtained in Example 1 has a significant antibacterial effect against agricultural pathogenic *Amorphophallus konjac*.

[0046] Example 5: Antibacterial effect against *Actinidia kiwifruit*, an agricultural pathogen.

[0047] The bacterial strain used, *Pseudomonas syringae* pv. *actinidiae*, was activated on beef extract peptone agar plates at 37°C for 24 h. Colonies were picked and added to MH broth, and cultured with shaking for 6 h. The bacterial suspension was then diluted to 1.0 × 10⁻⁶. 4 ~1.0×10 5 CFU / mL was prepared for use. Citrus amycin A was dissolved in DMSO and diluted with MH 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 medium was added; for the positive group, 100 μL of positive control solution and 100 μL of bacterial suspension were added; for the test group, 100 μL of different concentrations of citrus amycin A solution and 100 μL of bacterial suspension were added; and for the growth group, 100 μL of MH medium and 100 μL of bacterial suspension were added. After incubation at 37℃ 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 10 The inhibition rate of 12.5 μg / mL of citrinum A against *Actinidia kiwifruit* bacterial canker was 49.8%, and that of 6.25 μg / mL was 17.8%. Therefore, the MIC of citrinum A against *Actinidia kiwifruit* bacterial canker was calculated to be 11.8 μg / mL. Citrinum A showed significant antibacterial effect against this agriculturally pathogenic bacterium.

[0048] In summary, this application presents a novel citrinin derivative, citrione A, with a novel tetracyclic skeleton and a rare spirocyclic skeleton. It is a polymer formed from quinone-type citrinin and 2-hexane-1,3-diene-2,5-dimethylfuranone. Subsequent studies revealed that citrione A significantly inhibited the growth of four human pathogenic bacteria, with MICs ranging from 22.5 to 67.3 μg / mL. It also showed significant inhibitory effects against two agricultural pathogenic bacteria, with an MIC of 9.1 μg / mL against *Actinidia konjac* soft rot and 11.8 μg / mL against *Actinidia kiwifruit* bacterial canker. Such compounds can be used in the preparation of antibacterial drugs and precursors for agricultural antibiotics, laying a material foundation for further research on antibacterial efficacy.

[0049] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A fungal citrinin derivative, citranaxol A, characterized by, The structural formula of the citromycetin A is 2. The citromycetin A of claim 1 is applied in the preparation of drugs for resisting human pathogenic bacteria, wherein the human pathogenic bacteria are standard strains of Staphylococcus aureus, clinical strains of Staphylococcus aureus, Bacillus subtilis and Clostridium sporogenes, and the MIC of the citromycetin A is 22.5-67.3 μg / mL.

3. The citromycetin A of claim 1 is applied in the prevention and treatment of S. costatum and Pseudomonas syringae pv. actinidiae.

4. A process for preparing the satrumamycin derivative satranidin A according to claim 1, characterized in that, The method comprises the following steps: (1): After the activation of G. coronata, seed liquid is formed and then fermented in solid culture medium; the preparation method of the solid culture medium is as follows: 70-130 g of rice, 80-190 mL of water, 0.02-1.0 g of K2HPO4, soaked overnight and then autoclaved at 100-120 ℃ for 10-50 min; (2): After the ethyl acetate cold extraction of the fermentation product obtained in step (1), the crude extract is obtained by vacuum concentration; (3): After the impurities in the crude extract in step (2) are removed by silica gel column chromatography, the target sub-component is separated and purified by macroporous resin column chromatography and high performance liquid chromatography; the elution gradient of the macroporous resin column chromatography is 10%-100% ethanol-water; the target sub-component is 50%-90% ethanol-water; the eluent of the high performance liquid chromatography is acetonitrile and water, the volume ratio of acetonitrile to water is 40%-80% acetonitrile-water, and the detection wavelength is 210 nm.

Images