Phenol derivative in mangrove endophytic fungus and preparation method and application thereof

Abstract

The present application provides a kind of phenol derivatives in mangrove endophytic fungi and its preparation method and application, the compound has neuroprotective activity to the cell viability of H2O2 induced nerve cell (HT22) oxidative damage, can be used to prepare neuroprotective drug.The structural formula of its compound is as follows:

Description

Technical Field

[0001] This invention relates to the field of plant endophytic microbial technology, specifically to a phenol derivative in mangrove endophytic fungi, its preparation method, and its application. Background Technology

[0002] Mangrove ecosystems are located in the intertidal zone of tropical and subtropical coasts, a unique transitional zone between land and sea. Mangrove plants, as part of this ecosystem, have a unique growth environment: year-round exposure to strong sunlight, periodic submersion of their roots by seawater, and periodic hypoxia. This harsh yet unique environment leads to a rich abundance of specialized endophytic fungi adapted to this environment. These fungi can produce many lead compounds with excellent biological activity, unique chemical structures, and novel mechanisms of action. Studies have reported that mangrove fungi can produce a variety of novel and diverse compounds, mainly including alkaloids, quinones, macrolides, anthrones, peptides, polyketides, isocoumarins, and heteroterpenes. Many of these compounds exhibit good biological activity, including antitumor, antibacterial, antiviral, shrimp lethality, inhibition of acetylcholinesterase, inhibition of α-glucosidase, inhibition / promotion of angiogenesis, inhibition of calcium and potassium ion channels, antioxidant effects, and neuroprotective properties, providing a large number of drug progeny for new drug development.

[0003] Lumnitzera littorea (Jacq.) Voigt., belonging to the genus Lumnitzera in the family Combretaceae, is listed in the "National Key Protected Wild Plants List (First Batch)" (Level II) approved by the State Council of China on August 4, 1999. According to literature reports, in recent decades, scientists from various countries have isolated various types of compounds from Lumnitzera plants, including flavonoids, terpenes, sterols, and polyketides, and some of these compounds have shown good cytotoxic and anti-inflammatory activities.

[0004] The scarcity of *Prunus armeniaca* plant resources has hindered scientific research on it. In recent years, research on endophytic fungi in plants has increased both domestically and internationally, resulting in the isolation of various types of compounds, including polyketides, phenolic acids, indoles, and alkaloids. Some of these compounds exhibit good cytotoxic and anti-inflammatory activities. This invention aims to isolate and study the secondary metabolites of endophytic fungi in *Prunus armeniaca*, and, through effective extraction processes, discover compounds with good bioactivity, providing an important technical foundation for fully realizing the medicinal value of *Prunus armeniaca*. Summary of the Invention

[0005] The technical problem to be solved by this invention is as follows: In view of this, this invention proposes a phenol derivative compound from mangrove endophytic fungi, its preparation method and application.

[0006] To address the above problems, the technical solution provided by this invention is as follows:

[0007] A phenol derivative from a mangrove endophytic fungus, wherein the compound is a phenol derivative and its structural formula is as follows: .

[0008] To further explain, the compound was isolated and extracted from the secondary metabolites obtained by fermentation culture of the endophytic fungus Aspergillus terreus HT-1 from the mangrove plant Lumnitzera littorea.

[0009] To further clarify, the endophytic fungus *Aspergillus terreus* HT-1, belonging to the mangrove plant *Prunus cerasifera*, was deposited on December 27, 2021, at [location missing]. Wuhan, Hubei city Wuhan The university's China Center for Type Culture Collection (CCTCC) has assigned this strain the accession number CCTCC M 20211677 HT-1.

[0010] To further explain, the fermentation culture of the endophytic fungus Aspergillus terreus HT-1 of the mangrove plant Prunus rubra involves inoculating the seed culture of the endophytic fungus Aspergillus terreus HT-1 into potato dextrose aqueous liquid culture medium, and then incubating it at a constant temperature of 28°C for 28 days. The crude secondary metabolites are obtained by soaking in organic solvents, and then separated and purified to obtain phenol derivative compounds.

[0011] A method for preparing phenol derivative compounds from mangrove endophytic fungi includes the following steps:

[0012] S1. Activation of the strain: Take out the preserved endophytic fungus Aspergillus terreus HT-1 and place it in an incubator for pre-culture for 24 hours. Then, transfer it to a sterile PDA (potato dextrose agar) plate for activation and incubate it in an incubator for 2-4 days.

[0013] S2. Seed culture preparation: Take the endophytic fungus Aspergillus terreus HT-1 cells the size of a broad bean from step S1, inoculate them into sterile potato dextrose aqueous liquid culture medium, and culture them in a constant temperature shaker at 28℃ for 2-3 days to obtain the seed culture.

[0014] S3. Preparation of extract: The cultured seed liquid is transferred to sterile potato glucose aqueous liquid culture medium in a sterile laminar flow hood using a pipette. It is then fermented at a constant temperature of 28℃ for 28 days to obtain the fermentation product. The product is then extracted 2-3 times by soaking in an organic solvent and concentrated under reduced pressure to obtain crude extract.

[0015] S4. Chromatographic Separation: The crude extract was subjected to silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate (100:0-0:100) and chloroform-methanol (10:1-0:100). The eluents were collected, combined, and concentrated according to TLC analysis to obtain 19 fractions Fr.1~Fr.19. Fraction Fr.10 was isolated by a combination of ODS column chromatography, Sephadex LH-20 column chromatography, and HPLC. Its structure was identified by a combination of physicochemical analysis and modern spectroscopic methods such as NMR and MS. A phenol derivative compound, 4-Hydroxy-3-(3-methylbut-2-enyl)benzaldehyde, was isolated from the fermentation product of this strain.

[0016] To further explain, in step S1, the endophytic fungus Aspergillus terreus HT-1 was stored in a PDB (potato dextrose solution)-30% glycerol medium at -80°C; the constant temperature of the incubator was 28°C.

[0017] To further explain, the potato glucose aqueous liquid culture medium formulation in steps S2 and 3 includes 300 g / L potato extract powder and 20 g / L glucose.

[0018] To further explain, in step S3, the fermentation product is mycelium and bacterial suspension, which are extracted 2-3 times by soaking in an equal volume of ethyl acetate as an organic solvent. The extracts are combined and concentrated under reduced pressure to obtain crude extract.

[0019] Application of a phenol derivative compound from a mangrove endophytic fungus, wherein the compound is used in the preparation of neuroprotective drugs.

[0020] The application of a phenol derivative compound from a mangrove endophytic fungus, specifically its use in the preparation of neuroprotective drugs.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The phenol derivative compounds proposed in the present invention are derived from the secondary metabolites of the endophytic fungus Aspergillus terreus HT-1 of the mangrove plant Lumnitzera littorea. These compounds are obtained by using the secondary metabolites of the endophytic fungus Aspergillus littorea as raw materials, through artificial fermentation culture, solvent extraction, and separation by silica gel column chromatography, reversed-phase silica gel column chromatography, and recrystallization.

[0022] This invention achieves the isolation of phenolic derivatives from the secondary metabolites of the endophytic fungus *Aspergillus terreus* HT-1 in *Lumnitzera littorea*. Furthermore, it enables the enrichment of greater quantities of these phenolic derivatives through artificial fermentation. This method is simple, feasible, and inexpensive. Under the fermentation conditions of the endophytic fungus *Aspergillus terreus* HT-1 in *Lumnitzera littorea* according to this invention, phenolic derivatives in the fermentation product are obtained. The preparation method is simple, and the obtained phenolic derivatives have a high content.

[0023] Meanwhile, neuroprotective activity tests showed that the compound obtained in this invention can enhance the cell viability of H2O2-induced oxidative damage in HT22 neurons. At a concentration of 200 µM, it can increase the cell survival rate of H2O2-induced oxidative damage in HT22 neurons from 44.06% to 80.96%. It can be used in the preparation of neuroprotective drugs. Attached Figure Description

[0024] Figure 1 The proton NMR spectrum of the compound of the present invention. 1 H NMR (400 MHz, Acetone-d6);

[0025] Figure 2 The carbon spectrum of the compound of the present invention. 13 C NMR (100 MHz, Acetone-d6). Detailed Implementation

[0026] The present invention is illustrated below with reference to examples, but is not intended to limit the invention. Any simple substitutions or modifications made to the present invention by those skilled in the art are within the scope of the technical solutions protected by this invention.

[0027] Example 1:

[0028] The endophytic fungus Aspergillus terreus HT-1 of the mangrove plant *Prunus cerasifera* of this invention was provided by the strain bank of the Key Laboratory of Tropical Medicinal Resources Chemistry, Ministry of Education, Hainan Normal University. The mangrove plant *Prunus cerasifera* was collected from the roots of the endangered plant *Prunus cerasifera* in the Tielugang Mangrove Nature Reserve, Sanya City, Hainan Province. After isolation and purification, the strain was identified as *Aspergillus terreus* by morphology and molecular biology.

[0029] The bacterial strain was sent to a sequencing company for identification (testing company: Qingdao Pengxiang Biotechnology Co., Ltd.). The obtained base sequence was compared with the GenBank database for similarity, and then the BLAST program was used to search for homologous sequences for comparison. The HT-1 strain was identified as Aspergillus terreus, with a sequence similarity of 99%.

[0030] Example 2: Preparation of phenol derivative compounds

[0031] 1. Endophytic fungi Aspergillus terreus Fermentation culture of HT-1:

[0032] (1) Culture of strains: Aspergillus terreus The HT-1 strain was stored in PDB-glycerol medium at -80°C. The PDB-glycerol medium consisted of: 300 g / L potato, 20 g / L glucose, and 30% glycerol.

[0033] (2) Activation of strains: The strains stored at -80℃ are activated. Aspergillus terreus HT-1 was pre-cultured in a 28℃ incubator for 24 hours, and then inoculated onto PDA plates in a sterile laminar flow hood for activation, and cultured in a 28℃ incubator for 3 days.

[0034] (3) Fermentation culture: endophytic fungi the size of broad beans are fermented. Aspergillus terreus HT-1 cells were inoculated into sterile potato liquid culture medium and cultured in a shaker at 28°C for 3 days to obtain seed culture. 5 ml of the seed culture was transferred into sterilized potato liquid culture medium in a sterile laminar flow hood using a pipette and cultured statically at 28°C for 28 days to obtain fermentation product.

[0035] The potato glucose liquid culture medium consisted of 300 g / L potato, 20 g / L glucose, and 30 g / L sea salt. A total of 200 Erlenmeyer flasks with a capacity of 1000 mL were used for inoculation, with each flask containing 300 mL of fermentation broth.

[0036] 2. Extraction of crude extract:

[0037] The mycelial liquid and mycelium after fermentation were extracted three times with ethyl acetate, concentrated under reduced pressure, and the ethyl acetate extracts were combined to obtain a total of 23.6 g of crude extract.

[0038] 3. Isolation and purification of compounds:

[0039] The crude extract (23.6 g) was subjected to silica gel column chromatography (200-300 mesh) with gradient elution of petroleum ether-ethyl acetate (100:0-0:100) and chloroform-methanol (10:1-0:100), collecting approximately 0.5 L of fractions each time. The fractions were combined and concentrated according to TLC analysis to obtain 19 fractions Fr.1–Fr.19. Fraction Fr. 10 was further eluted using reversed-phase C18 column chromatography (MeOH:H2O: 20:80-100:0) to obtain 16 subfractions Fr.10-1–Fr.10-16. Fr.10-7 was purified by silica gel column chromatography (200-300 mesh) with a gradient elution of petroleum ether-ethyl acetate (100:0-0:100), collecting fractions in increments of approximately 0.25 L. TLC analysis yielded five fractions (Fr.10-7-1-Fr.10-7-5). Fr.10-7-3 was further purified by semi-preparative high-performance liquid chromatography (CH3OH / H2O, v / v, 56:44; flow rate: 3.0 ml / min) to obtain the compound (3.2 mg). The structural formula of the compound is shown below: .

[0040] Example 3: Structural Identification of Phenol Derivatives

[0041] The chemical structure of the compound obtained in Example 2 was determined using modern structural identification techniques such as spectrometry and MS.

[0042] The structural assessment data is as follows:

[0043] Compound: It is a yellow oil that is readily soluble in organic solvents such as acetone and methanol. 1 H NMR (400 MHz, Acetone-d6)δH 7.67 (d, J = 2.0 Hz, 1H, H-2), 7.00 (d, J = 8.0 Hz, 1H, H-5), 7.63 (dd,J = 2.4, 8.4 Hz,1H, H-6), 9.82 (s, 1H, H-7), 3.37 (d, J = 4.2 Hz, 2H, H-1'), 5.36 (m, J = 5.4 Hz, 1H, H-2'), 1.74 (d, J = 0.8 Hz, 3H, H-4'), 1.73 (s, 3H,H-5'),. 13C NMR (101 MHz, Acetone-d6)δC 129.8 (C-1), 131.8 (C-2), 130.6 (C-3), 161.6 (C-4), 116.0 (C-5), 130.7 (C-6), 191.2 (C-8), 28.7 (C-1'), 122.8 (C-2'), 133.5 (C-3'), 25.9 (C-4'), 17.8 (C-5').

[0044] Example 4: Neuroprotective activity test of phenol derivative compounds

[0045] 1. Neuroprotective activity test

[0046] 1.1 Experimental Materials

[0047] Cells: Mouse hippocampal neurons (HT22, Beijing Beina Chuanglian Biotechnology Research Institute);

[0048] Cell culture medium: 90% DMEM-H (Hyclone) + 10% FBS (GIBCO);

[0049] Reagent: MTT (Sigma-Aldrich (Shanghai) Trading Co., Ltd.).

[0050] 1.2 Experimental Methods

[0051] 1.2.1 Grouping

[0052] The study was divided into five groups: blank group, model group (H2O2 damage group), and drug group (5, 10, 50, 100, 200µM).

[0053] 1.2.2 Effects of compounds on cell viability after H2O2-induced oxidative damage to nerve cells

[0054] The effect of compounds on the cell viability of H2O2-induced oxidative damage in neural cells (HT22) was detected using the MTT assay. Cells were seeded in 96-well plates at 1 x 10³ cells / well, with a volume of 200 μL per well. After overnight incubation, the cells were used for experiments. The culture medium was discarded, and the cells were gently washed once with PBS. The cells were randomly divided into 7 groups. The control group and the model group were added to medium containing 1% FBS, while the drug groups were added to medium containing 5, 10, 50, 100, and 200 µM of 1% FBS, respectively, with 100 µL per well. After 24 h of incubation, the control group was added to 100 µL of 1% FBS, and the other groups were added to medium containing 1% FBS (final concentration of H2O2 100 µM). After 24 h of incubation, 20 μL of MTT solution was added to each well, and the cells were incubated for another 4 h. The liquid was discarded, and 150 μL of DMSO was added. The absorbance (OD value) of each well was measured at 490 nm. Repeat the experiment three times. Each group was set to have three replicates.

[0055] 1.2.3 Data Processing

[0056] Analysis of variance was performed using SPSS software, and the results are expressed as mean ± standard deviation (±s).

[0057] 1.3 Activity Test Results

[0058] The test results are shown in Table 1 below:

[0059] Table 1. Effects of compounds on cell viability after H2O2-induced oxidative damage to HT22 cells ( (n=6)

[0060]

[0061] After H2O2-induced oxidative damage for 24 h, HT22 cell viability was significantly reduced (P<0.01). Different concentrations of the drug (5, 10, 50, 100, 200 µM) could improve cell viability to varying degrees after incubation for 24 h, as shown in Table 1. Compound 1 had a better anti-oxidative damage effect.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing phenolic derivatives from mangrove endophytic fungi, characterized in that, It comprises the following steps: S1, strain activation: the preserved endophytic fungus Aspergillus terreus HT-1 is taken out and pre-cultured in an incubator for 24 hours, then transferred to a sterile PDA plate for activation and cultured in the incubator for 2-4 days; S2, seed liquid preparation: the endophytic fungus Aspergillus terreus HT-1 in step S1 is inoculated into sterile potato glucose aqueous liquid medium, cultured at 28℃ in a constant temperature incubator for 2-3 days to obtain a seed liquid; S3, extract preparation: the seed liquid is inoculated into sterile potato glucose aqueous liquid medium, and fermented at 28℃ for 28 days to obtain a fermentation product, which is soaked with an organic solvent for 2-3 times, concentrated under reduced pressure to obtain a crude extract; S4, chromatographic separation: the crude extract is subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate 100:0-0:100 and chloroform-methanol 10:1-0:100 in sequence to obtain a phenol derivative from mangrove endophytic fungus; the phenol derivative compound has the following structural formula: ; the compound is isolated by fermentation of Aspergillus terreus HT-1, an endophytic fungus of mangrove Lumnitzera littorea; the endophytic fungus Aspergillus terreus HT-1 of the mangrove Lumnitzera littorea is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 20211677 HT-1.

2. A process for the preparation of phenolic derivatives from mangrove endophytic fungi as claimed in claim 1, wherein, In step S1, the endophytic fungus Aspergillus terreus HT-1 is preserved in a PDB-30% glycerol medium at-80℃, and the constant temperature culture temperature of the incubator is 28℃.

3. A process for the preparation of phenolic derivatives from mangrove endophytic fungi as claimed in claim 2, wherein, The potato glucose aqueous liquid medium in steps S2 and S3 comprises potato infusion powder 300g / L and glucose 20g / L.

4. The method of claim 3, wherein the phenolic derivative is prepared from a mangrove endophytic fungus, and the fungus is Mangrove endophytic fungus 1. In step S3, the fermentation product is mycelium and bacterial suspension, which is soaked with an equal volume of ethyl acetate as an organic solvent for 2-3 times, the extract is combined and concentrated under reduced pressure to obtain a crude extract.

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