Mangrove sediment-derived fungus and application thereof

Abstract

The application provides a mangrove sediment-derived fungus and application thereof. Penicillium The preservation number of the sp. UJNMF0740 strain is CGMCC No.40521; and the above-mentioned Penicillium fungus and fermentation products thereof can be used for producing antibacterial drugs. The application also provides an indole diterpene compound with antibacterial effect produced by the above-mentioned Penicillium fungus, which has a good application prospect in developing drugs or antibacterial additives with antibacterial effect.

Description

Technical Field

[0001] This invention belongs to the field of marine biology, specifically relating to a Penicillium fungus and its antibacterial applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus, also known as Staphylococcus aureus, is a common Gram-positive bacterium and a zoonotic pathogen. It is a globally recognized foodborne pathogen, widely distributed in aquatic products (fish, shrimp, etc.), meat products (pork, poultry, beef, mutton, etc.), and dairy products. It can cause various diseases in humans and animals, such as membranous colitis, pneumonia, pericarditis, and other localized purulent infections, and even systemic infectious diseases such as sepsis and septicemia, seriously threatening human and animal safety. With the emergence of methicillin-resistant Staphylococcus aureus and other drug-resistant strains, Staphylococcus aureus has become a global health problem.

[0003] Mangroves refer to the woody biological communities of tidal wetlands, mainly composed of evergreen shrubs or trees, growing in the upper intertidal zone of tropical and subtropical coasts. They represent a unique ecosystem transitioning from land to sea. Mangrove ecosystems are periodically submerged by seawater, exhibiting high dynamic salinity levels, large organic matter reserves, and high nutrient cycling rates. They possess characteristics of both marine and terrestrial environments, yet also have their own unique features, fostering abundant microbial resources. Fungi are a major group of microorganisms in mangroves, possessing the biosynthetic potential to produce various novel compounds and a rich variety of physiologically active substances. They are an important source of active metabolites with medicinal value, such as antibacterial and antitumor agents. Therefore, screening mangrove sediment fungi with antibacterial effects is of great significance for the development of novel antibacterial drugs. Summary of the Invention

[0004] In response to the urgent need for antibacterial drugs, this invention provides a Penicillium fungus ( Penicillium sp.UJNMF0740), whose fermentation products have antibacterial effects.

[0005] Another objective of this invention is to provide an indole diterpenoid compound with antibacterial activity isolated from Penicillium fungal ferments and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A Penicillium fungus Penicillium sp. UJNMF0740 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40521.

[0008] A Penicillium fungus PenicilliumApplication of *Penicillium* sp. UJNMF0740 and its fermentation products in the preparation of antibacterial drugs. Penicillium After being cultured, sp. UJNMF0740 can produce fermentation products with antibacterial activity, which can be used to produce drugs or additives with antibacterial activity.

[0009] Penicillium fungi Penicillium The indole diterpenoids produced by sp. UJNMF0740 have the structures shown in formula (I): Formula (I) The preparation method of the above-mentioned indole diterpenoid compounds includes the following steps: (1) Penicillium fungi Penicillium Fermentation of sp. UJNMF 0740 yielded fermentation product; (2) The crude fermentation extract was obtained from the fermentation product obtained in step (1) by ethanol extraction and ethyl acetate extraction; (3) The fermented crude extract in step (2) is separated in the following manner: (3-1) The fermentation crude extract was eluted with dichloromethane:methanol at a volume ratio of 1:0-0:1 through a 100-200 mesh normal phase silica gel column to obtain fractions Fr.1-Fr.9; (3-2) Fraction Fr.2 was eluted with a gradient of petroleum ether:acetone at a volume ratio of 1:0-0:1 through a 200-300 mesh normal phase silica gel column to obtain fractions Fr.2.1-Fr.2.7; Fr.2.3 was eluted with dichloromethane:methanol at a volume ratio of 1:1 through a Sephadex LH-20 column to obtain fractions Fr.2.3.1-Fr.2.3.5; Fr.2.3.2 was eluted with methanol:water at a volume ratio of 80:20 through a C18 column semi-preparative high performance liquid chromatography to obtain the compound shown in formula (I).

[0010] The present invention provides the use of the fermented crude extract in step (2), or the compound shown in formula (I), in the preparation of antibacterial drugs, pharmaceutical intermediates, and antibacterial additives.

[0011] This invention provides an antimicrobial drug or antimicrobial additive prepared from the fermented crude extract of step (2) or the compound shown in formula (I). The antimicrobial drug further includes medically acceptable excipients and / or other active ingredients to broaden the bactericidal spectrum or enhance the antimicrobial effect.

[0012] The present invention has the following advantages: The indole diterpenoid compounds of the present invention can be used to... PenicilliumIt was obtained by fermentation extraction and separation of sp. UJNMF0740, which can inhibit the growth of Staphylococcus aureus and has the potential for application in the preparation of antibacterial drugs or antibacterial additives.

[0013] Biological Preservation Information Penicillium sp. UJNMF 0740 was deposited on March 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 40521. Attached Figure Description

[0014] Figure 1 Fungi Penicillium Image of sp.UJNMF0740 plate culture; Figure 2 The main compound shown in formula (I) 1 H- 1 H COSY, HMBC information; Figure 3 The main NOESY information for the compound shown in formula (I); Figure 4 The ECD spectrum of the compound shown in formula (I) is shown below. Figure 5 for Penicillium Cytotoxic activity of the fermentation extract of sp. UJNMF0740 and the compound shown in formula (I). Detailed Implementation

[0015] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0016] Example 1: Isolation, purification and identification of UJNMF 0740 Rhizosphere soil samples from the mangrove forest of *Kandelia candel* in Dongzhai Port, Hainan Province, were collected and diluted using a serial dilution method to obtain a soil suspension. In a clean bench, 100 μL of this soil suspension was pipetted into an isolation medium and spread evenly using a spreader. The culture dish was then inverted and placed in a 28°C incubator for 1-2 weeks to observe for colony growth. The mycelia were then purified by streak plating onto PDA medium using a sterile bamboo stick. The resulting bacterial strain was obtained after isolation and purification. Penicillium sp. UJNMF0740, a typical culture image is shown below. Figure 1 As shown: Colonies are round with a white periphery and green hyphae. ITS rDNA detection indicates they are similar to those in Gene Bank. Penicillium svalbardense(NR_111508.1) has 99.08% homology and was identified as a Penicillium fungus.

[0017] Example 2: Fermentation and Crude Extract Preparation of UJNMF 0740 Preparation of seed culture medium: 200 mL potato extract, 20 g glucose, 30 g sea salt, and water to a final volume of 1 L. Pour the culture medium into 20 500 mL Erlenmeyer flasks, approximately 150 mL per flask, and autoclave at 121°C for 25 minutes.

[0018] Preparation method of rice fermentation medium: 80g rice, 0.4g yeast extract, 0.4g glucose, 120ml water, 3% sea salt. Place in 1L Erlenmeyer flasks, for a total of 150 flasks. Autoclave at 121℃ for 25 minutes and set aside.

[0019] Use a sterile bamboo skewer to pick out an appropriate amount of fungus. Penicillium The UJNMF 0740 strain was inoculated into seed culture medium and cultured on a shaker (200 rpm) at 28°C for 3 days to obtain seed liquid. Then, 10 mL of seed liquid was inoculated into a 1 L Erlenmeyer flask containing rice fermentation medium and cultured statically at 28°C for 30 days before the fermentation product was collected.

[0020] After 30 days of fermentation, the rice fermentation medium was transferred out and crushed. It was then soaked in 95% ethanol four times (each soaking lasted 6-7 days). The 95% ethanol was collected after each soaking and concentrated into a solution using a large rotary evaporator. Then, it was extracted with an equal amount of ethyl acetate for a total of five extractions. The extracts were combined and the solvent was evaporated to obtain approximately 35g of crude fermentation extract.

[0021] Example 3: Antibacterial activity of UJNMF 0740 fermented crude extract (1) Antibacterial zone The effects of UJNMF 0740 extract on the efficacy of paper disc diffusion method were determined. Staphylococcus aureus Antibacterial activity of ATCC25923: The above-mentioned bacterial strains were inoculated into LB liquid medium and cultured at 37°C on a shaker (180 rpm) for 24 hours. Then, the bacterial concentration of the culture was diluted to approximately 10% using the McFarland turbidimetric method with LB liquid medium. 8 CFU / mL is prepared for use; at 50-60℃, 200 μL of the above bacterial strain dilution is added to every 200 mL of sterile LB solid medium and then quickly poured into a sterile petri dish with a diameter of 9 cm. After the plate cools, a 6 mm sterile paper disc (containing 100 μg of the sample to be tested) is placed in the above plate and incubated at 37℃ for 18 hours, and the size of the inhibition zone is observed.

[0022] Experimental results show thatPenicillium The crude fermentation extract of sp. UJNMF0740 could inhibit [the growth of bacteria] at a concentration of 100 μg / paper disc. Staphylococcus aureus The growth of bacteria was inhibited by a 7.5 mm inhibition zone.

[0023] (2) IC 50 Measurement by Staphylococcus aureus ATCC25923 was used as the test strain. First, the strain was inoculated onto LB solid medium for resuscitation. Then, an appropriate amount of colonies was picked and inoculated onto liquid medium and cultured at 37°C and 160 rpm using a shaker. The cultured strain was then diluted to 10⁻⁶ using blank medium. 8 cfu / mL. 190 μL was pipetted into a 96-well plate, followed by 10 μL of DMSO solution containing the crude extract. The plate was incubated overnight at 37°C. The absorbance (OD value) was measured at 600 nm using a microplate reader, and the inhibition rate was calculated. Blank culture medium was used as a control.

[0024] Experimental results show that Penicillium sp.UJNMF0740 fermentation extract on S. aureus It has antibacterial activity, and its IC 50 The concentration was 73.2 μM, which has the effect of inhibiting... S. aureus Growth activity.

[0025] Example 4 Preparation of indole diterpenoids The fermentation crude extract was obtained according to the method in Example 2, and subjected to silica gel column chromatography (100-200 mesh) with gradient elution of dichloromethane:methanol (1:0-0:1 v / v). During elution, thin-layer chromatography silica gel plates were used for detection and combination of similar fractions, resulting in a total of 9 fractions (Fr.1-Fr.9).

[0026] Fr.2 was analyzed by silica gel column chromatography (200-300 mesh) with gradient elution of petroleum ether:acetone (1:0-0:1 v / v), followed by detection using thin-layer chromatography on silica gel plates, yielding 7 fractions (Fr.2.1-Fr.2.7). Fr.2.3 was analyzed by Sephadex LH-20 (dichloromethane:methanol = 1:1), yielding 5 fractions (Fr.2.3.1-Fr.2.3.5). Fr.2.3.2 was analyzed by semi-preparative high-performance liquid chromatography (methanol / water, 80:20 v / v, 3 mL / min) to obtain the compound (t) shown in formula (I). R = 12 min, 1.5 mg).

[0027] The compound shown in formula (I) is an amorphous white powder with an optical rotation of [α]. 25 D 15.7 (c 0.23, MeOH).

[0028] The isolated compounds were subjected to high-resolution mass spectrometry (HR-ESIMS). 1 H NMR, 13 C NMR, 2D 1 H- 1 HCl COSY, HSQC, and HMBC analyses determined the planar structure, and the configuration of the compound was further determined by NOESY and ECD spectra. 1 H and 13 The C NMR data are shown in Table 1, mainly... 1 H- 1 For information regarding H COSY and HMBC, please see [link / reference]. Figure 2 .

[0029] Table 1 shows the compounds represented by formula (I) in CDCl3. 1 H (600 MHz) and 13 C (150 MHz) NMR data The high-resolution mass spectrometry (HR-ESIMS) of the compound shown in formula (I) yielded a quasi-molecular ion peak at m / z of 598.3166 ([M + H]+, calculated 598.3163). Combined with the NMR spectral data, the molecular formula of the compound is deduced to be C. 37 H 43 NO6 has an unsaturation degree of 17. Analysis of the NMR data of this compound revealed a striking similarity to that of shearinine D (Xu M., 2007), indicating a similar structural fragment. The difference lies in the NMR spectra of shearinine D. C / δ H The hydroxymethyl carbon signal at 76.7 / 4.86 (CH-22) disappears in the compound shown in formula (I), while δ C A carbonyl signal appears at position 201.7, suggesting that the C-22 position in the compound shown in formula (I) has been converted to a carbonyl group. This is confirmed by related signals in the HMBC spectrum from H-23 to C-22 / C-24, and from H-20 to C-22. Figure 2 ).

[0030] NOE spectrum ( Figure 3 The relative configuration of the compound can be inferred from the correlations between H-16 and H-14β, H-16 and H3-33, H3-32 and H-17α, H3-32 and H-5α, H3-32 and H-15α, and H-14α and H-11. Then, by calculating (3... S 4R 7 S 9 R ,13 S ,16 S ,twenty three R )-1 and (3 S 4 R 7 S 9 R ,13 S ,16 S ,twenty three S The theoretical ECD spectrum of )-2, and the experimental ECD curve of the compound shown in formula (I) are consistent with that of 2. Figure 4 Its absolute configuration is determined to be 3. S 4 R 7 S 9 R ,13 S ,16 S ,twenty three S : Example 5 Antibacterial activity of compound (I) by Staphylococcus aureus ATCC25923 was used as the test strain. First, the strain was inoculated onto LB solid medium for resuscitation. Then, an appropriate amount of colonies was picked and inoculated onto liquid medium and cultured at 37°C and 160 rpm using a shaker. The cultured strain was then diluted to 10⁻⁶ using blank medium. 8 cfu / mL. 190 μL was pipetted into a 96-well plate, followed by 10 μL of DMSO solution containing a certain concentration of the compound. The plate was incubated overnight at 37°C. The absorbance (OD value) was measured at 600 nm using a microplate reader, and the inhibition rate was calculated. Blank culture medium was used as a control.

[0031] Experimental results show that the compound shown in formula (I) is effective against... S. aureus It has antibacterial activity, and its IC 50 The concentration is 51.0 μM, which has the effect of inhibiting... S. aureus Growth activity.

[0032] Example 6 Penicillium Cytotoxicity of fermentation extract of sp. UJNMF0740 and compounds represented by formula (I) PC12 cells were divided into groups of 1.5 × 10⁻⁶. 5Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. Control and experimental groups were set up. The experimental group was incubated with 100 μM of the test agent for 12 hours, while the control group was incubated with an equal volume of DMSO solution. Subsequently, 10 μL of MTT solution was added to each well, and the cells were incubated at 37°C for 4 hours. After removing the culture medium, 150 μL of DMSO was added to dissolve formaldehyde crystals, and the optical density (OD) was measured at 490 nm using a microplate reader.

[0033] Experimental results show that ( Figure 5 ), Penicillium The crude extract of sp.UJNMF0740 fermentation and the compound shown in formula (I) showed no significant cytotoxic activity against PC12 cells at 100 μM, and the compound shown in formula (I) showed certain proliferative activity against PC12 cells.

Claims

1. An indole diterpenoid compound, the structure of which is shown in formula (I): Formula (I).

2. A method for preparing the indole diterpenoid compound as described in claim 1, characterized in that, Includes the following steps: (1) Penicillium fungi Penicillium Fermentation of sp. UJNMF 0740 yielded fermentation product; (2) The crude fermentation extract was obtained from the fermentation product obtained in step (1) by ethanol extraction and ethyl acetate extraction; (3) The fermented crude extract in step (2) is separated in the following manner: (3-1) The fermentation crude extract was eluted with dichloromethane:methanol at a volume ratio of 1:0-0:1 through a 100-200 mesh normal phase silica gel column to obtain fractions Fr.1-Fr.9; (3-2) Fraction Fr.2 was eluted with a gradient of petroleum ether:acetone at a volume ratio of 1:0-0:1 through a 200-300 mesh normal phase silica gel column to obtain fractions Fr.2.1-Fr.2.7; Fr.2.3 was eluted with dichloromethane:methanol at a volume ratio of 1:1 through a Sephadex LH-20 column to obtain fractions Fr.2.3.1-Fr.2.3.5; Fr.2.3.2 was eluted with methanol:water at a volume ratio of 80:20 through a C18 column semi-preparative high performance liquid chromatography to obtain the compound shown in formula (I); Penicillium fungi Penicillium sp. UJNMF0740 has the accession number CGMCC No. 40521.

3. A drug with antibacterial activity, characterized in that, Contains an effective amount of the indole diterpenoid compound as an active ingredient as described in claim 1.

4. The drug according to claim 3, characterized in that, It also includes medically acceptable excipients.

5. The use of the indole diterpenoid compound of claim 1 in the preparation of a drug with antibacterial activity, characterized in that, The drug targets Staphylococcus aureus. Staphylococcus aureus .

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