A fungus derived from marine sediments and its applications

Abstract

This invention belongs to the field of biomedicine and provides a fungus derived from marine sediments and its applications. The fungus is a fungus of the genus Aspergillus. Aspergillus tanneir UJNMF0417, with accession number CGMCC No. 41647. The fermentation product of this *Aspergillus* fungus, after extraction with an organic solvent, yielded a crude extract with good neuroprotective activity. The indolepiperazine compounds provided by this invention can be... Aspergillus tanneir UJNMF0417 was obtained through fermentation extraction and isolation. It can improve 6-OHDA-induced PC-12 cell damage and has potential applications in the preparation of drugs for the prevention and treatment of PD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fungus of the genus Aspergillus and its applications. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people, mainly characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. As the disease progresses, the motor and non-motor symptoms of PD gradually worsen, impacting patients' daily lives and imposing a significant social and medical burden. In recent years, exogenous neurotoxin-induced neural cell models have become a fundamental tool in PD research, among which the 6-OHDA-induced PC-12 cell (rat adrenal pheochromocytoma cells) damage model has become an ideal model for in vitro basic research on drugs for the prevention and treatment of PD.

[0004] Due to various sedimentary processes, the seabed is often covered with a layer of sediment of varying thickness. Continuous material exchange occurs between the sediment and the bottom seawater, giving marine sediments both marine and terrestrial characteristics, as well as unique features, fostering rich microbial resources. Fungi are a major group of microorganisms in marine sediments, capable of producing a wide variety of physiologically active substances and possessing the potential to generate various novel compounds. They are an important source of active metabolites with medicinal value, such as anti-inflammatory and anti-tumor agents. Screening metabolites from their fermentation products that can improve PC-12 cell damage is of great significance for developing drugs with therapeutic and preventative effects against Parkinson's disease. Summary of the Invention

[0005] In response to the urgent need for drugs to treat Parkinson's disease, this invention provides a fungus of the genus Aspergillus (… Aspergillus tanner UJNMF0417, whose fermentation product has the effect of improving 6-OHDA-induced PC-12 cell damage, can be used in the research and development of drugs for the treatment and prevention of Parkinson's disease.

[0006] Another objective of this invention is to provide an indolepiperazine compound isolated from Aspergillus fungal fermentation products that can improve 6-OHDA-induced PC-12 cell damage and its preparation method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A type of Aspergillus fungus Aspergillus tanneirUJNMF0417 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41647.

[0009] The above-mentioned Aspergillus fungi Aspergillus tanneir UJNMF0417 can be used in the production of drugs or pharmaceutical intermediates for the prevention and treatment of Parkinson's disease. The Aspergillus fungus in question... Aspergillus tanneir UJNMF0417 can produce fermentation products after culturing. These fermentation products can improve 6-OHDA-induced PC-12 cell damage and can be used as drugs or lead compounds for the development of Parkinson's disease.

[0010] A fermentation product of the aforementioned Aspergillus fungus, through fermentation Aspergillus tanneir UJNMF0417 was obtained.

[0011] The present invention also provides extracts of the above-mentioned Aspergillus fungi.

[0012] The extract is obtained by extracting the fermentation product with an organic solvent, wherein the organic solvent is ethanol and ethyl acetate; preferably, the concentration of ethanol is 90%-95%.

[0013] The extract is obtained by extracting the fermentation product with 90%-95% ethanol, extracting the extract with ethyl acetate, removing the solvent from the extract, and then separating it by D101 macroporous resin column chromatography, eluting with 30%, 50%, 75%, and 95% ethanol, retaining the second fraction, and removing the solvent.

[0014] The extract is obtained by extracting the fermentation product with 90%-95% ethanol, followed by extraction with ethyl acetate. After removing the solvent, the extract is separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol, retaining the second fraction. The second fraction is then subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane / methanol at a volume ratio of 1:0-0:1, retaining the seventh fraction, and the solvent is removed to obtain the final product.

[0015] The extract is obtained by extracting the fermentation product with 90%-95% ethanol, followed by extraction with ethyl acetate. After removing the solvent, the extract is separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol, retaining the second fraction. The second fraction is then subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane / methanol at a volume ratio of 1:0-0:1, retaining the seventh fraction. Finally, the extract is obtained by passing it through a Sephadex LH-20 column, retaining the third fraction, and removing the solvent.

[0016] The extract is obtained by extracting the fermentation product with 90%-95% ethanol, followed by extraction with ethyl acetate. After solvent removal, the extract is separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol, retaining the second fraction. The second fraction is then subjected to normal-phase silica gel column chromatography with a dichloromethane / methanol gradient elution phase of 1:0-0:1 (v / v), retaining the seventh fraction. This fraction is then passed through a Sephadex LH-20 column, retaining the third fraction. The third fraction is then passed through a reverse-phase C20 column. 18 Liquid chromatography was performed by elution with a methanol / water gradient of 30:70-90:10 (v / v), retaining the third fraction and removing the solvent.

[0017] An indolepiperazine compound has the structure shown in formula (I):

[0018]

[0019] Formula (I).

[0020] The preparation method of the above-mentioned indolepiperazine compounds includes the following steps:

[0021] (1) Aspergillus spp. Aspergillus tanneir UJNMF0417, obtained fermentation product;

[0022] (2) The fermentation product obtained in step (1) was successively extracted with ethanol and ethyl acetate to obtain a crude extract;

[0023] (3) The crude extract obtained in step (2) was separated by D101 macroporous resin column chromatography and eluted with 30%, 50%, 75% and 95% ethanol to obtain four fractions Fr.1-Fr.4 in sequence.

[0024] Fr.2 was subjected to normal-phase silica gel column chromatography with a mobile phase of dichloromethane / methanol with a volume ratio of 1:0-0:1 as the gradient elution phase, and eight fractions, Fr.2.1-Fr.2.8, were obtained sequentially.

[0025] Fraction Fr.2.7 was passed through a Sephadex LH-20 chromatography column to obtain five fractions, Fr.2.7.1 to Fr.2.7.5.

[0026] Fr.2.7.3 via inverted C 18 Liquid chromatography, using a methanol / water gradient elution at a volume ratio of 30:70-90:10, yielded fractions Fr.2.7.3.1-Fr.2.7.3.5 sequentially;

[0027] Fr.2.7.3.3 was subjected to preparative high-performance liquid chromatography (HPLC) with a mobile phase of methanol / water at a volume ratio of 60:40 and a flow rate of 3.0 mL / min, with a t-retention capacity of [missing value]. RThe effluent was obtained by removing the mobile phase after 27 min of effluent.

[0028] The concentration of ethanol in step (2) is 90%-95%.

[0029] The present invention provides the use of the above-mentioned Aspergillus fungi, fermentation products, extracts, or compounds as shown in formula (I) in the preparation of medicaments for treating Parkinson's disease and pharmaceutical intermediates.

[0030] A composition comprising the compound shown in formula (I) above and its pharmaceutically acceptable salt. Preferably, the salt is a hydrochloride or sulfate formed from the -NH- group of an indolepiperazine compound.

[0031] The present invention has the following advantages:

[0032] The Aspergillus fungi provided by this invention are derived from nature, have high safety, and the crude extract obtained after sequential extraction with ethanol and ethyl acetate from the fermentation product exhibits good neuroprotective activity. The indolepiperazine compounds provided by this invention can be... Aspergillus tanneir UJNMF0417 was obtained through fermentation extraction and isolation. It can improve 6-OHDA-induced PC-12 cell damage and has potential applications in the preparation of drugs for the prevention and treatment of PD.

[0033] Biological Preservation Information

[0034] Aspergillus tanneir It was deposited on December 2, 2024, 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. 41647. Attached Figure Description

[0035] Figure 1 Fungi Aspergillus tanneir Image of UJNMF0417 plate culture;

[0036] Figure 2 The main target compound 1 H- 1 H COSY, HMBC information;

[0037] Figure 3 The main NOESY information for the target compound;

[0038] Figure 4 Fungi Aspergillus tanneir Cytotoxicity of UJNMF0417 crude extract against PC-12 cells;

[0039] Figure 5The cytotoxicity of indolepiperazine compounds to PC-12 cells;

[0040] Figure 6 Fungi Aspergillus tanneir Neuroprotective effect of crude extract of UJNMF0417 on PC-12 cells damaged by 6-OHDA;

[0041] Figure 7 This study investigated the neuroprotective effects of indolepiperazine compounds on PC-12 cells damaged by 6-OHDA. Detailed Implementation

[0042] The present invention will be further described below with reference to embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods in the embodiments employ conventional techniques in the art, and the experimental reagents are all commercially available.

[0043] Example 1: Screening, isolation, purification and identification of bacterial strains

[0044] Sediment samples were collected from Shangchuan Island, Taishan City, Guangdong Province in May 2019. The bacterial strain UJNMF0417 was obtained after isolation and purification. A typical culture image is shown below. Figure 1 As shown: Colonies are round with yellow hyphae. ITS rDNA testing identified it as... Aspergillus tanneir Its ITS rDNA is Gene Bank No. ON707164. The strain was deposited and numbered CGMCC No. 41647.

[0045] Example 2 Preparation of fermentation product

[0046] 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.

[0047] Rice culture medium preparation method: 80g rice, 0.4g yeast extract, 0.4g glucose, 120mL water, 3% sea salt. Place in a 1L Erlenmeyer flask and autoclave at 121℃ for 25 minutes.

[0048] Use a sterile bamboo skewer to pick up an appropriate amount Aspergillus tanneir The UJNMF0417 strain was inoculated into the above seed culture medium and cultured on a shaker (200 rpm) at 28°C for 3 days to obtain the seed liquid. Then, 10 mL of the seed liquid was inoculated into a 1 L Erlenmeyer flask containing rice culture medium and cultured statically at 28°C for 30 days before the fermentation product was collected.

[0049] After fermenting for 30 days, the rice culture 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 an aqueous solution using a large rotary evaporator. Then, it was extracted with an equal volume of ethyl acetate five times. After further concentration using a rotary evaporator, a crude extract was obtained.

[0050] Example 3 Preparation of indolepiperazine compounds

[0051] 90 g of the fermentation extract obtained in Example 2 was subjected to macroporous resin D101 column chromatography and eluted with 30%, 50%, 75%, and 95% ethanol to obtain a total of 4 fractions (Fr.1-Fr.4).

[0052] Fr.2 was eluted by silica gel column chromatography with a gradient of dichloromethane:methanol (1:0-0:1). During elution, thin-layer chromatography silica gel plates were used for detection and similar fractions of compounds were combined, resulting in a total of 8 fractions (Fr.2.1-Fr.2.8).

[0053] Fr.2.7 was analyzed by Sephadex LH-20 gel column chromatography combined with thin-layer chromatography silica gel plate, yielding 5 fractions (Fr.2.7.1-Fr.2.7.5). Fr.2.7.3 was analyzed by RP-C... 18 Column chromatography was performed, eluting with methanol / water (30:70-90:10). The eluted components were then analyzed using a thin-layer chromatography silica gel plate, and concentrated to obtain five fractions (Fr.2.7.3.1-Fr.2.7.3.5). Fr.2.7.3.3 was separated by semi-preparative high-performance liquid chromatography (methanol / water, v / v 60:40, 3.0 mL / min) to obtain the target compound (t). R = 27 min, 6.6 mg), is a light pink oil.

[0054] High-resolution mass spectrometry (HR-ESIMS) was used to perform high-resolution mass spectrometry on the compound. 1 H NMR, 13 C NMR, 2D 1 H- 1 H COSY and HMBC analyses determined the planar structure, and the relative configuration of the compound was determined by combining the NOESY correlation signal. 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 For information related to NOESY, please see [link / reference]. Figure 3 .

[0055] Table 1. Target compounds in CDCl3 1 H (600 MHz) and 13 C (150 MHz) NMR data

[0056]

[0057] Structural identification: Based on the quasi-molecular ion peaks given by ESIMS mass spectrometry. m / z 404.1946 ([M + Na]) + (Calculated value 404.1945), combined with NMR data, the molecular formula of this compound is inferred to be C. 22 H 27 N3O3. 1 1H spectral data (see Table 1) show that this compound contains an active hydrogen atom ( d H 5.74), 3 aromatic protons ( d H 7.21, 7.19, 7.31), 3 olefin protons ( d H 7.20, 6.18, 7.11), 3 methyl signals ( d H 1.42, 1.40, 3.25). 13 C10 NMR and DEPT135 spectra show the presence of 22 carbons, including 3 methyl carbons (C10 NMR and DEPT135 spectra). d C 25.8, 25.8, 50.7), 4 methylene carbons ( d C 29.1, 28.5, 22.8, 45.6) 8 methylene carbons ( d C 124.5, 118.5, 123.2, 111.0, 55.0, 59.4, 127.8, 138.6) and 7 seasonal carbon signals ( d C 111.0, 124.0, 131.5, 137.7, 169.4, 165.4, 75.4).

[0058] By comparing with literature data, combined with 1 H- 1 H COSY and HMBC spectra ( Figure 2 This compound is similar to aspergillinine D, the main difference being the C-4 side chain. In the HMBC spectrum, -OC H 3 ( d H 3.25) and C-20 ( d C 75.4), H-21 ( d H 1.42) and H-22 ( d H 1.40) and C-19 ( d C 138.6) and H-5 ( d H 7.21) and C-18 ( d C The relevant signals of 138.6) and 1 H- 1 H-18 in the H COSY spectrum ( d H 7.20) and H-19 ( d H The relevant signal from 6.18 indicates that the compound is connected to 4-methoxy-4-methylpent-2-ene at the C-4 position, rather than the 2-methylpent-2-ene in aspergillinine D; in the NOESY spectrum, H-9 ( d H 4.32) and H-12 ( d H 4.02) Related Notes: H-9 and H-12 are located on the same side of the diketopiperazine ring and are both in an upright position. Therefore, the planar structure and relative configuration of this compound are determined as follows:

[0059] .

[0060] Example 4 Safety Evaluation

[0061] Rat adrenal pheochromocytoma cells (PC-12) were divided into groups of 1.0 × 10⁻⁶ cells. 5 Cells 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 groups were treated with crude extract or the indolepiperazine compound DMSO solution obtained in Example 3 to final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL and 25 μM, 50 μM, 100 μM, respectively, and incubated for 12 hours (the control group received 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, and the optical density (OD) was measured at 490 nm using a microplate reader.

[0062] The results are as follows Figure 4 , 5 As shown, fungi Aspergillus tanneirCompounds isolated from the crude extract and fermentation product of UJNMF0417 showed no significant cytotoxic activity against PC-12 cells.

[0063] Example 5 Evaluation of neuroprotective activity

[0064] Using 6-OHDA-induced PC-12 cell damage as a model, the fungi obtained in Example 2 were evaluated. Aspergillus tanner The anti-PD activity of the crude extract of UJNMF0417 and the compounds isolated in Example 3 was evaluated. Cell viability was determined using the MTT assay to assess the neuroprotective activity of the analytes.

[0065] Experimental results are as follows Figure 6 , 7 As shown, at a test concentration of 100 μg / mL, the fungi... Aspergillus tanner The crude extract of UJNMF0417 can reduce 6-OHDA-induced PC-12 cell damage; after treatment with indolepiperazine compounds at a concentration of 50 μM, the survival rate of PC-12 cells was significantly increased compared with the damaged group (6-OHDA), indicating that the crude extract and indolepiperazine compounds can reduce 6-OHDA-induced PC-12 cell death and have certain neuroprotective effects, which can be used for the development of drugs for the treatment or prevention of Parkinson's disease.

[0066] The above description is merely an 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 principle of this application should be included within the scope of the claims of this application.

Claims

1. A strain of Aspergillus fungus ( Aspergillus tanneir UJNMF0417, characterized in that, The preservation number is CGMCC No. 41647.

2. An extract of the Aspergillus fungus of claim 1, characterized in that, selected from Fermented Aspergillus fungus (Aspergillus oryzae) Aspergillus tanneir ) UJNMF0417 was obtained by fermentation; then extracted by 90-95% ethanol and ethyl acetate in turn; or Fermented Aspergillus fungus Aspergillus tanneir ) UJNMF0417 obtain fermentation product; fermentation product is extracted by 90%-95% ethanol, extract is extracted by ethyl acetate, extract is removed solvent, and then separated by D101 macroporous resin column chromatography, eluted by 30%, 50%, 75%, and 95% ethanol, second fraction is reserved, and solvent is removed to obtain; or, Fermented Aspergillus sp. fungi Aspergillus tanneir ) UJNMF0417 to obtain a fermentation product; the fermentation product is extracted with 90-95% ethanol, the extract is extracted with ethyl acetate; after removing the solvent from the extract, the extract is separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol, and the second fraction is retained; the second fraction is subjected to normal-phase silica gel column chromatography with dichloromethane / methanol in a volume ratio of 1:0-0:1 as the gradient elution mobile phase, the seventh fraction is retained, and the solvent is removed to obtain; or, Fermented Aspergillus sp. fungi Aspergillus tanneir UJNMF0417 to obtain fermentation products; fermentation products are extracted with 90%-95% ethanol, and the extract is extracted with ethyl acetate; after removing the solvent from the extract, the extract is separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol, and the second fraction is reserved; the second fraction is subjected to normal-phase silica gel column chromatography with dichloromethane / methanol in a volume ratio of 1:0-0:1 as the gradient elution mobile phase, and the seventh fraction is reserved; then, the third fraction is reserved by passing through a Sephadex LH-20 chromatography column, and the solvent is removed to obtain; or, Fermented Aspergillus sp. fungi Aspergillus tanneir UJNMF0417 to obtain fermentation product; fermentation product extracted with 90-95% ethanol, extract liquid extracted with ethyl acetate; after removing solvent of extract liquid, separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, 95% ethanol, reserved second fraction; second fraction subjected to normal phase silica gel column chromatography with dichloromethane / methanol (1:0-0:1, by volume) as gradient elution mobile phase, reserved seventh fraction; then passed through Sephadex LH-20 chromatography column, reserved third fraction; third fraction passed through reverse phase C 18 liquid chromatography with methanol / water (30:70-90:10, by volume) gradient elution, reserved third fraction, removed solvent to obtain.

3. An indolizine compound, the structure of which is shown in formula (I): Formula (I).

4. A method for preparing an indolepiperazine compound as described in claim 3, characterized in that, comprising the following steps: (1) Aspergillus spp. fungi ( Aspergillus tanneir UJNMF0417 was used to obtain the fermentation product; (2) The fermentation obtained in step (1) is extracted with ethanol and ethyl acetate in sequence to obtain a crude extract; (3) The crude extract obtained in step (2) is separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol to obtain four fractions Fr.1-Fr.4; Fr.2 is subjected to normal phase silica gel column chromatography with dichloromethane / methanol in a gradient of 1:0-0:1 by volume ratio as the mobile phase to obtain eight fractions Fr.2.1-Fr.2.8; Fraction Fr.2.7 is subjected to Sephadex LH-20 chromatography column to obtain five fractions Fr.2.7.1-Fr.2.7.5; Fr. 2.7.3 was isolated by reverse phase C 18 Fr. 2.7.3.1 - Fr. 2.7.3.5 fractions were obtained by gradient elution with methanol / water 30:70 to 90:10 in volume ratio by liquid chromatography. Fr. 2.7.3.3 was purified by preparative high performance liquid chromatography, using a mobile phase of methanol / water 60:40 by volume at a flow rate of 3.0 mL / min, and the effluent was collected for 27 min, and the mobile phase was removed; R to give the title compound. MS (M+1): 184.

1. The Aspergillus fungus (Aspergillus sp.) Aspergillus tanneir ) UJNMF0417 has a deposit number of CGMCC No. 41647.

5. Use of the Aspergillus fungus of claim 1, the extract of claim 2, or the indolizine compound of claim 3 in the preparation of a medicament for treating Parkinson's disease.

6. A composition containing the indolizine compound of claim 3 and pharmaceutically acceptable salts thereof.

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