Marine sediment-derived fungus and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides a marine sediment-derived fungus and application thereof, the fungus is Aspergillus tanner UJNMF0417, and the preservation number is CGMCC No.41647. The invention also provides a preparation method of the marine sediment-derived fungus, and the marine sediment-derived fungus has the advantages that the marine sediment-derived fungus is prepared from the marine sediment-derived fungus, the marine sediment-derived fungus and the application of the marine sediment-derived fungus. A crude extract obtained after the fermentation product of the aspergillus fungus is extracted by an organic solvent has good neuroprotective activity. The indole piperazine compound # imgabs0 # provided by the invention can be obtained by fermentation, extraction and separation of Aspergillus tanner UJNMF0417, can improve PC-12 cell injury induced by 6-OHDA, and has application potential in the aspect of preparing medicines for preventing and treating PD (Parkinson's Disease).

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an Aspergillus fungus and an application thereof. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Parkinson's disease (PD) is a common neurodegenerative disease in the elderly, characterized by progressive degeneration of dopaminergic neurons in the substantia nigra. As the disease progresses, the motor and non-motor symptoms of Parkinson's disease will gradually worsen, which will affect the patient's daily life on the one hand, and also bring huge social and medical burdens on the other hand. In recent years, the neural cell model induced by exogenous neurotoxins has become a basic means of studying Parkinson's disease, among which the 6-OHDA-induced PC-12 cell (rat adrenal pheochromocytoma cell) injury model has become an ideal model for in vitro basic research on drugs for the prevention and treatment of Parkinson's disease.

[0004] Due to various sedimentary processes, the seabed is often covered with a layer of sediments of varying thickness. There is a constant exchange of materials between the sediments and the bottom seawater, which makes the marine sediments have both the properties of the ocean and the land and have their own unique characteristics, and are rich in microbial resources. Fungi are a major group of microorganisms in marine sediments. They can produce a wide variety of physiologically active substances and have the potential to produce a variety of new compounds. They are an important source of active metabolites with medicinal value such as anti-inflammatory and anti-tumor. Screening out metabolites that can improve PC-12 cell damage from their fermentation products is of great significance for the development of drugs that can treat and prevent Parkinson's disease. Summary of the invention

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

[0006] Another object of the present invention is to provide an indole piperazine compound isolated from an Aspergillus fungus fermentation product and capable of improving 6-OHDA-induced PC-12 cell damage and a preparation method thereof.

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

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

[0009] Aspergillus fungi Aspergillus tanneir UJNMF0417 can be used to produce drugs or drug intermediates for preventing and treating Parkinson's disease. Aspergillus tanneir UJNMF0417 can produce fermentation products after cultivation. The fermentation products can improve 6-OHDA-induced PC-12 cell damage and can be used as drugs or lead compounds for the development of prevention and treatment of Parkinson's disease.

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

[0011] The present invention also provides an extract of the Aspergillus fungus.

[0012] The extract is obtained by extracting the fermentation product with an organic solvent, and the organic solvent is ethanol and ethyl acetate; preferably, the concentration of the 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, separating the extract with 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 a fermentation product extracted with 90%-95% ethanol, and the extract is extracted with ethyl acetate; after the solvent is removed from the extract, it 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 a mobile phase of dichloromethane / methanol with a volume ratio of 1:0-0:1 as a gradient elution, and the seventh fraction is retained and the solvent is removed to obtain the extract.

[0015] The extract is a fermentation product extracted with 90%-95% ethanol, and the extract is extracted with ethyl acetate; after the solvent is removed from the extract, it 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 a mobile phase of dichloromethane / methanol with a volume ratio of 1:0-0:1 as a gradient elution, and the seventh fraction is retained; then it is passed through a Sephadex LH-20 chromatography column, the third fraction is retained, and the solvent is removed to obtain the product.

[0016] The extract is a fermentation product extracted with 90%-95% ethanol, and the extract is extracted with ethyl acetate; after the solvent is removed from the extract, it 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 a mobile phase of dichloromethane / methanol with a volume ratio of 1:0-0:1 as a gradient elution, and the seventh fraction is retained; then it is passed through a Sephadex LH-20 chromatography column, and the third fraction is retained; the third fraction is separated by reverse phase C 18 Liquid chromatography was performed with a gradient elution of methanol / water in a volume ratio of 30:70-90:10, and the third fraction was retained and the solvent was removed to obtain the product.

[0017] An indole piperazine compound, the structure of which is shown in formula (I): Formula (I).

[0018] The preparation method of the above-mentioned indole piperazine compound comprises the following steps: (1) Fermentation of Aspergillus fungi Aspergillus tanneir UJNMF0417, obtained fermentation; (2) extracting the fermented product obtained in step (1) with ethanol and ethyl acetate in sequence to obtain a crude extract; (3) The crude product obtained in step (2) was separated by D101 macroporous resin column chromatography, and eluted with 30%, 50%, 75%, and 95% ethanol to obtain four parts, Fr.1-Fr.4, respectively; Fr.2 was subjected to normal phase silica gel column chromatography using dichloromethane / methanol in a volume ratio of 1:0-0:1 as a gradient elution mobile phase to obtain eight fractions, Fr.2.1-Fr.2.8; The fraction Fr.2.7 was passed through a Sephadex LH-20 chromatography column to obtain five fractions Fr.2.7.1-Fr.2.7.5 in sequence; Fr.2.7.3 By inverting C 18 Liquid chromatography, with a gradient elution of methanol / water in a volume ratio of 30:70-90:10, yielded fractions Fr.2.7.3.1-Fr.2.7.3.5; Fr.2.7.3.3 was purified by preparative HPLC using methanol / water in a volume ratio of 60:40 as the mobile phase at a flow rate of 3.0 mL / min with a retention time of t R The effluent at 27 min was obtained by removing the mobile phase.

[0019] The concentration of ethanol in step (2) is 90%-95%.

[0020] The present invention provides a use of the Aspergillus fungus, fermentation product, extract, or compound represented by formula (I) in the preparation of drugs and drug intermediates for treating Parkinson's disease.

[0021] A composition containing the compound represented by formula (I) and a pharmaceutically acceptable salt thereof. Preferably, the salt is a hydrochloride or sulfate formed by -NH- in an indole piperazine compound.

[0022] The present invention has the following advantages: The Aspergillus fungus provided by the present invention is derived from nature and has high safety. The crude extract of the fermented product after sequential extraction with ethanol and ethyl acetate has good neuroprotective activity. The indole piperazine compound provided by the present invention can be Aspergillus tanneir UJNMF0417, obtained by fermentation extraction and separation, can improve 6-OHDA-induced PC-12 cell damage and has application potential in the preparation of drugs for the prevention and treatment of PD.

[0023] Biological deposit information Aspergillus tanneir , deposited on December 2, 2024 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC No.41647. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For fungi Aspergillus tanneir UJNMF0417 plate culture picture; Figure 2 The main target compound 1 H- 1 H COSY, HMBC information; Figure 3 The main NOESY information of the target compound; Figure 4 For fungi Aspergillus tanneir The crude extract of UJNMF0417 was cytotoxic to PC-12 cells; Figure 5 It is the cytotoxicity of indolepiperazine compounds to PC-12 cells; Figure 6 For fungi Aspergillus tanneir The neuroprotective effect of crude extract of UJNMF0417 on 6-OHDA-damaged PC-12 cells; Figure 7 The neuroprotective effect of indolepiperazine compounds on 6-OHDA-damaged PC-12 cells. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the examples and drawings, but the present invention is not limited by the following examples. The experimental methods in the examples, unless otherwise specified, all adopt conventional techniques in the art, and the experimental reagents are all purchased commercially.

[0026] Example 1 Screening, separation, purification and identification of bacterial strains In May 2019, sediment samples were collected from Shangchuan Island, Taishan City, Guangdong Province. The samples were separated and purified to obtain the strain UJNMF0417. The typical culture picture is as follows: Figure 1 As shown: The colony is round and the hyphae are yellow. ITS rDNA detection identified it as Aspergillus tanneir Its ITS rDNA is Gene Bank No.ON707164. The strain is deposited and numbered as CGMCC No.41647.

[0027] Example 2 Preparation of fermented product Preparation method of seed culture medium: 200 mL of potato extract, 20 g of glucose, 30 g of sea salt, dilute to 1 L with water. Pour the culture medium into 20 500 mL conical flasks, about 150 mL per flask, sterilize at 121℃ with high pressure steam for 25 minutes, and set aside.

[0028] Preparation method of rice culture medium: 80g rice, 0.4g yeast extract, 0.4g glucose, 120mL water, 3% sea salt. Place in a 1L Erlenmeyer flask, sterilize at 121℃ with high pressure steam for 25 minutes, and set aside.

[0029] Use a sterile bamboo stick to pick up an appropriate amount Aspergillus tanneir The UJNMF0417 strain was inoculated into the above seed culture medium and cultured at 28°C in a shaker (200 rpm) for 3 days to obtain the seed solution. Then, 10 mL of the seed solution was inoculated into a 1 L Erlenmeyer flask containing rice culture medium using a pipette. After static culture at 28°C for 30 days, the fermentation product was collected.

[0030] The rice culture medium after fermentation for 30 days was transferred out and crushed, and then soaked in 95% ethanol for 4 times (single soaking time was 6-7 days). The 95% ethanol after each soaking was collected and concentrated using a large rotary evaporator to obtain an aqueous solution. The aqueous solution was then extracted with an equal amount of ethyl acetate for five times, and the crude extract was concentrated using a rotary evaporator.

[0031] Example 3 Preparation of Indole Piperazine Compounds 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).

[0032] Fr.2 was subjected to silica gel column chromatography gradient elution with dichloromethane:methanol (1:0-0:1). During the elution, a thin layer chromatography silica gel plate was used for detection and the fractions with similar compounds were combined to obtain a total of 8 fractions (Fr.2.1-Fr.2.8).

[0033] Fr.2.7 was purified by Sephadex LH-20 gel column and detected by thin layer chromatography silica gel plate to obtain 5 fractions (Fr.2.7.1-Fr.2.7.5). Fr.2.7.3 was purified by RP-C 18 Column chromatography was performed, eluted with methanol / water (30:70-90:10), and then the eluted components were detected by thin layer chromatography silica gel plate and concentrated to obtain 5 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 = 27min, 6.6 mg), as a light pink oil.

[0034] The compounds were subjected to high-resolution mass spectrometry (HR-ESIMS), 1 H NMR, 13 C NMR, 2D 1 H- 1 The planar structure was determined by H COSY and HMBC analysis, and the relative configuration of the compound was determined by combining NOESY related signals. 1 H and 13 C NMR data are shown in Table 1. The main 1 H- 1 For more information on H COSY and HMBC, see Figure 2 For more information about NOESY, see Figure 3 .

[0035] Table 1 Target compounds in CDCl 3 In 1 H (600 MHz) and 13 C (150 MHz) NMR data Structural identification: Based on the quasi-molecular ion peaks given by mass spectrometry (ESIMS) m / z 404.1946 ([M + Na] +, calculated value 404.1945), combined with NMR data, it is inferred that the molecular formula of the compound is C 22 H 27 N 3 O 3 . 1 H spectrum data (see Table 1) show that the compound contains an active hydrogen ( 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 C NMR and DEPT135 spectra showed that it contained 22 carbon atoms, including 3 methyl carbon atoms ( d C 25.8, 25.8, 50.7), 4 methylene carbons ( d C 29.1, 28.5, 22.8, 45.6) 8 methyl carbons ( d C 124.5, 118.5, 123.2, 111.0, 55.0, 59.4, 127.8, 138.6) and 7 quaternary carbon signals ( d C 111.0, 124.0, 131.5, 137.7, 169.4, 165.4, 75.4).

[0036] By comparing with literature data, combined with 1 H- 1 H COSY and HMBC spectra ( Figure 2 ), which is similar to aspergillinine D, with the main difference being the side chain at C-4. 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 ( dH 7.21) and C-18 ( d C 138.6) and related signals 1 H- 1 H COSY spectrum of H-18 ( d H 7.20) and H-19 ( d H 6.18), indicating that the compound C-4 is connected to 4-methoxy-4-methylpent-2-ene, rather than 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 explanation H-9 and H-12 are on the same side of the diketopiperazine ring and are both upright. Therefore, the planar structure and relative configuration of the compound are determined as follows: .

[0037] Example 4 Safety Evaluation Rat adrenal pheochromocytoma cells (PC-12) were cultured at a rate of 1.0 × 10 5 The cells were seeded at a density of 100 μL / well in a 96-well plate and incubated at 37°C with 5% CO 2 The cells were cultured in a constant temperature incubator for 24 hours. A control group and an experimental group were set up. The experimental group was added with crude extract or DMSO solution of indole piperazine compounds obtained in Example 3 to a final concentration of 25 μg / mL, 50 μg / mL, 100 μg / mL and 25 μM, 50 μM, 100 μM, respectively, and the cells were incubated for 12 hours (the control group was added with an equal volume of DMSO solution). Subsequently, 10 μL of MTT solution was added to each well, incubated at 37°C for 4 hours, the culture medium was removed, 150 μL of DMSO was added, and the optical density (OD) was measured at 490 nm using a microplate reader.

[0038] The results are as follows Figure 4 , 5 As shown, fungi Aspergillus tanneir The compounds isolated from the crude extract and fermentation product of UJNMF0417 had no obvious cytotoxic activity against PC-12 cells.

[0039] Example 5 Evaluation of neuroprotective activity The fungus obtained in Example 2 was evaluated using 6-OHDA-induced PC-12 cell injury as a model. Aspergillus tannerAnti-PD activity of crude extract of UJNMF0417 and the compound isolated in Example 3. In the experiment, the MTT method was used to determine the cell survival rate to evaluate whether the test substance has neuroprotective activity.

[0040] The experimental results are as follows Figure 6 , 7 As shown, at a test concentration of 100 μg / mL, 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 injury group (6-OHDA), indicating that the crude extract and indolepiperazine compounds can reduce 6-OHDA-induced PC-12 cell death, have a certain neuroprotective effect, and can be used in the development of drugs for the treatment or prevention of Parkinson's disease.

[0041] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

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

2. A fermentation product and extract of Aspergillus fungus as claimed in claim 1.

3. The extract according to claim 2, characterized in that Selected from Fermentation Aspergillus tanneir UJNMF0417 to obtain the fermentation product; then extracting it with 90%-95% ethanol and ethyl acetate in sequence; or Fermentation Aspergillus tanneir UJNMF0417 to obtain a fermentation product; the fermentation product is extracted with 90%-95% ethanol, the extract is extracted with ethyl acetate, the extract is removed from the solvent and then separated by D101 macroporous resin column chromatography, eluted with 30%, 50%, 75%, and 95% ethanol, the second fraction is retained, and the solvent is removed to obtain; or, Fermentation Aspergillus tanneir UJNMF0417 obtains a fermentation product; the fermentation product is extracted with 90%-95% ethanol, and the extract is extracted with ethyl acetate; the extract is separated by D101 macroporous resin column chromatography after the solvent is removed, and eluted with 30%, 50%, 75%, and 95% ethanol, retaining the second fraction; the second fraction is subjected to normal phase silica gel column chromatography with a mobile phase of dichloromethane / methanol in a volume ratio of 1:0-0:1 as a gradient elution, retaining the seventh fraction, and removing the solvent to obtain; or, Fermentation Aspergillus tanneir UJNMF0417 obtains the fermentation product; the fermentation product is extracted with 90%-95% ethanol, and the extract is extracted with ethyl acetate; the extract is separated by D101 macroporous resin column chromatography after the solvent is removed, and eluted with 30%, 50%, 75%, and 95% ethanol, retaining the second fraction; the second fraction is 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 a gradient elution, and the seventh fraction is retained; then the second fraction is passed through a Sephadex LH-20 chromatography column, the third fraction is retained, and the solvent is removed to obtain; or, Fermentation Aspergillus tanneir UJNMF0417 obtained fermentation products; the fermentation products were extracted with 90%-95% ethanol, and the extract was extracted with ethyl acetate; the extract was separated by D101 macroporous resin column chromatography after the solvent was removed, and eluted with 30%, 50%, 75%, and 95% ethanol, and the second fraction was retained; the second fraction was 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 was retained; then the second fraction was subjected to Sephadex LH-20 chromatography column, and the third fraction was retained; the third fraction was separated by reverse phase C 18 Liquid chromatography was performed with a gradient elution of methanol / water in a volume ratio of 30:70-90:10, and the third fraction was retained and the solvent was removed to obtain the product.

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

5. A method for preparing an indole piperazine compound as claimed in claim 4, characterized in that: The following steps are involved: (1) Fermentation of Aspergillus fungi Aspergillus tanneir UJNMF0417, obtained fermentation; (2) extracting the fermented product obtained in step (1) with ethanol and ethyl acetate in sequence to obtain a crude extract; (3) The crude product 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; Fr.2 was subjected to normal phase silica gel column chromatography using dichloromethane / methanol in a volume ratio of 1:0-0:1 as a gradient elution mobile phase to obtain eight fractions from Fr.2.1 to Fr.2.8; 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; Fr.2.7.3 By inverting C 18 Liquid chromatography, with a gradient elution of methanol / water in a volume ratio of 30:70-90:10, afforded fractions Fr.2.7.3.1-Fr.2.7.3.5; Fr.2.7.3.3 was purified by preparative HPLC using methanol / water in a volume ratio of 60:40 as the mobile phase at a flow rate of 3.0 mL / min with a retention time of t R The effluent at 27 min was obtained by removing the mobile phase; The Aspergillus fungus Aspergillus tanneir The deposit number of UJNMF0417 is CGMCC No.41647.

6. Use of the Aspergillus fungus according to claim 1, the fermentation product and extract according to claim 2 or 3, or the indolepiperazine compound according to claim 4 in the preparation of drugs and drug intermediates for treating Parkinson's disease.

7. A composition comprising the indolepiperazine compound as claimed in claim 4 and a pharmaceutically acceptable salt thereof.

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