A phenolic compound from a mangrove-derived fungus and its preparation method

Abstract

This invention relates to a phenolic compound from mangrove-derived fungi and its preparation method, wherein the phenolic compound has the structure shown in compound 1:

Description

Technical Field

[0001] This invention belongs to the field of secondary metabolites of mangrove fungi, specifically relating to phenolic compounds from mangrove plant-derived fungi and their preparation methods. Background Technology

[0002] Mangrove plants grow in the tropical and subtropical intertidal zone, where the environment is characterized by high pressure, high salinity, and low oxygen. This environment enables their endophytic fungi to have unique metabolic pathways, resulting in the ability to produce compounds with novel structures and diverse biological activities. Their metabolites have various medicinal values, including antibacterial, antitumor, immunomodulatory, and enzyme-inhibiting properties, making them a potential resource for the development of microbial drugs. Therefore, mangrove endophytic fungi will become one of the important resources for new drug research and development.

[0003] This invention utilizes artificial culture and fermentation to obtain secondary metabolites with significant antibacterial activity from endophytic fungi of mangrove plants. This method is environmentally friendly and sustainable, effectively addressing key issues such as drug source in drug development, thus possessing unique advantages. The invention is derived from *Dothiorella* sp. ML002. Summary of the Invention

[0004] The fungus "Dothiorella sp. ML002" described in this invention was isolated from the mangrove plant *Melia azedarach*, which was collected by the inventors in August 2015 from the Dongzhai Port Mangrove Nature Reserve in the South China Sea, Hainan Province. Morphological and molecular biological identification of *Dothiorella sp. ML002* revealed that this strain belongs to the genus *Dothiorella*. The sequence of the ITS region of this fungus has been submitted to NCBI (GenBank accession No. MK131322). The fungus *Dothiorella sp. ML002* described in this invention has been disclosed in the inventor's previous research paper "Bioorganic Chemistry, 85, (2019), 382–385, Bioactive cytosporone derivatives isolated from the mangrove-derived fungus *Dothiorella sp. ML002*", and has been deposited at the Key Laboratory of Tropical Medicinal Resources Chemistry of the Ministry of Education, where the applicant is located. The public can obtain the fungus *Dothiorella sp. ML002* described in this invention by following the method described in the inventor's aforementioned research paper, or by purchasing it from the Key Laboratory of Tropical Medicinal Resources Chemistry of the Ministry of Education, where the applicant is located. The applicant promises that the Key Laboratory of Tropical Medicinal Resources Chemistry of the Ministry of Education will provide the public with the fungus *Dothiorella sp. ML002* described in this invention for 20 years from the date of this application.

[0005] This invention provides a phenolic compound or a pharmaceutically acceptable salt thereof, characterized in that the phenolic compound has the structure shown in compound 1:

[0006]

[0007] This invention provides a method for simultaneously preparing phenolic compounds 1 and 2, characterized by comprising the following steps:

[0008] (1) Inoculate the fungus Dothiorella sp.ML002 into the fermentation medium and incubate it at 26-28℃ for 21-28 days to obtain the fermentation product;

[0009] (2) Extract the fermentation product obtained in step (1) with 1-2 times the volume of ethyl acetate 2-4 times, combine the ethyl acetate phases and concentrate under reduced pressure to obtain the extract.

[0010] (3) The extract obtained in step (2) was subjected to vacuum silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient, and the extract was divided into seven fractions according to polarity. Fraction 1 was obtained from gradients 100:0 to 90:10, and fractions 80:20 to 70:10 were obtained from gradients 80:20 to 70:10. Fraction 2 was obtained by elution at a ratio of 0:30, fraction 3 by elution at a ratio of 60:40, fraction 4 by elution at a ratio of 50:50, fraction 5 by elution at a ratio of 40:60–30:70, fraction 6 by elution at a ratio of 20:80–10:90, and fraction 7 by elution at a ratio of 0:100. Fraction 4 was first subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate in a ratio of 8:1–3:1, eluting for 4–5 column volumes. After concentration under reduced pressure, it was then subjected to Sephadex chromatography. LH-20 gel column chromatography was performed using a mixed solvent of petroleum ether:CHCl3:MeOH = 2:1:1 as the eluent. After eluting for 4-5 column volumes, the solution was concentrated under reduced pressure and then prepared by high performance liquid chromatography (HPLC). The chromatographic column was a Waters C18, 9.4 × 250 mm, 7 μm, with a flow rate of 2 mL / min and a mobile phase of MeOH:H2O = 80:20. Compounds 1 and 2 were obtained.

[0011] The proportions of the eluent or mobile phase are all volume ratios; the fermentation culture medium is a solid rice culture medium (the formula is 70 ml of water and 2.2 g of natural sea salt per 60 g of rice).

[0012] Another embodiment of the present invention provides a crude extract of the fungus Dothiorella sp. ML002, characterized in that the preparation method of the crude extract includes the following steps:

[0013] (1) Inoculate the fungus Dothiorella sp.ML002 into the fermentation medium and incubate it at 26-28℃ for 21-28 days to obtain the fermentation product;

[0014] (2) Extract the fermentation product obtained in step (1) with 1-2 times the volume of ethyl acetate 2-4 times, combine the ethyl acetate phases and concentrate under reduced pressure to obtain the extract, which is the crude extract.

[0015] The fermentation medium is a solid rice culture medium (the formula is 70ml of water and 2.2g of natural sea salt per 60g of rice).

[0016] The present invention provides an anti-Candida albicans drug, characterized in that the above-mentioned phenolic compounds 1 and / or 2, or their pharmaceutically acceptable salts or the above-mentioned crude extracts are used as active ingredients.

[0017] The anti-Candida albicans drugs provided by the present invention also include other anti-Candida albicans drugs; they may also include pharmaceutically acceptable carriers or excipients.

[0018] Another embodiment of the invention provides the use of the phenolic compounds 1 and / or 2, or pharmaceutically acceptable salts thereof, or the crude extracts thereof, in the preparation of anti-Candida albicans medicaments.

[0019] In this invention, the term "pharmaceutically acceptable salt" refers to the addition salt of a nontoxic inorganic or organic acid and / or base, see "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201–217. Attached Figure Description

[0020] Figure 1 It is compound 1 1 H NMR spectrum;

[0021] Figure 2 It is compound 1 13 C NMR spectrum;

[0022] Figure 3 This is the 135-DEPT plot of compound 1;

[0023] Figure 4 This is the HMQC diagram of compound 1;

[0024] Figure 5 This is the COSY diagram of compound 1;

[0025] Figure 6 This is the HMBC diagram of compound 1;

[0026] Figure 7 This is the HR-ESI-MS chromatogram of compound 1. Detailed Implementation

[0027] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0028] Example 1

[0029] (1) Preparation of fermentation medium: The formula is to add 60g of rice, 70ml of water and 2.2g of natural sea salt to each conical flask. Incubate at 120℃ for 25–30 minutes.

[0030] The fungus Dothiorella sp. ML002 was inoculated into the fermentation medium and cultured statically at 26-28℃ for 28 days to obtain the fermentation product.

[0031] (2) The fermentation product obtained in step (1) was extracted three times with 2 times the volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to obtain the extract.

[0032] (3) The extract obtained in step (2) was subjected to vacuum silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient, and the extract was divided into seven fractions according to polarity. Fraction 1 was obtained from gradients 100:0 to 90:10, and fractions 80:20 to 70:10 were obtained from gradients 80:20 to 70:10. Fraction 2 was obtained by elution at a ratio of 0:30, fraction 3 by elution at a ratio of 60:40, fraction 4 by elution at a ratio of 50:50, fraction 5 by elution at a ratio of 40:60–30:70, fraction 6 by elution at a ratio of 20:80–10:90, and fraction 7 by elution at a ratio of 0:100. Fraction 4 was first subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate in a ratio of 8:1–3:1, eluting for 4–5 column volumes. After concentration under reduced pressure, it was then subjected to Sephadex chromatography. LH-20 gel column chromatography was performed using a mixed solvent of petroleum ether:CHCl3:MeOH = 2:1:1 as the eluent. After eluting for 4-5 column volumes, the solution was concentrated under reduced pressure and then prepared by high performance liquid chromatography (HPLC). The chromatographic column was a Waters C18, 9.4 × 250 mm, 7 μm, with a flow rate of 2 mL / min and a mobile phase of MeOH:H2O = 80:20. Compound 1 (5.0 mg) and compound 2 (3.6 mg) were obtained.

[0033]

[0034] The NMR structural confirmation data of compound 1 are as follows:

[0035] Table 1 Compound 1 1 H(400MHz)and 13 C(100MHz) NMR data (CDCl3)

[0036]

[0037]

[0038] Compound 1: Light brown powder; UV (MeOH)λmax(logε)221(3.12),230(3.11),265(2.90),296(3.40)nm; IR (KBr)ν max 3289,1735,1602,1462,1365,1230cm -1 ; 1 H and 13C NMR data(CDCl3)see Table 1; HR-ESI-MS m / z 321.1340[MH] - (calcd for C 17 H 21 O6,321.1338).

[0039] Compound 2 1 H / 13 The C NMR results are consistent with those of dothiorelone E (Phytochem. Lett., 22, 219 (2017)).

[0040] Example 2 Antibacterial Test

[0041] The compounds 1-2 and crude extracts of the present invention were tested for antibacterial activity against five pathogenic bacteria: Staphylococcus aureus (ATCC25923), S. albus (ATCC8032), Bacillus cereus (ATCC14579), Escherichia coli (ATCC35218), Vibrio parahaemolyticus (ATCC17749), and one pathogenic fungus, Candida albicans (ATCC10231), according to the microplate analysis method described in the literature (Pierce CG; Uppuluri P.; Teistan AR; Wormley Jr.FL; Mowat E.; Ramage G.; Lopez-ribot J.L. Nat. Protoc. 2008, 3, 1494-1500). During the initial screening, compounds 1-2 and the crude extract showed antibacterial activity against Candida albicans (ATCC 10231) at a concentration of 100 μg / mL. In particular, compound 2 and the crude extract showed significant antibacterial activity. After further dilution testing, the final MIC value of compound 2 against ATCC 10231 was 6.25 μg / mL, the MIC value of the crude extract against ATCC 10231 was 25.0 μg / mL, and the MIC value of the positive control drug ketoconazole was 3.12 μg / mL.

Claims

1. A method for simultaneously preparing phenolic compounds 1 and 2, characterized in that... Includes the following steps: (1) Inoculate the fungal strain Dothiorella sp. ML002 into the fermentation medium and incubate it at 26-28℃ for 21-28 days to obtain the fermentation product; (2) Extract the fermentation product obtained in step (1) with 1-2 times the volume of ethyl acetate 2-4 times, combine the ethyl acetate phases and concentrate under reduced pressure to obtain the extract; (3) The extract obtained in step (2) was subjected to vacuum silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:

100. Two column volumes were collected for each gradient, and the extract was divided into 7 fractions according to polarity. Fraction 1 was obtained by elution of gradients 100:0 to 90:10, and fractions 80:20 to 70:10 were obtained by elution of gradients 80:20 to 70:

10. Fraction 2 was obtained by elution at a gradient of 0:30, fraction 3 by elution at a gradient of 60:40, fraction 4 by elution at a gradient of 50:50, fraction 5 by elution at a gradient of 40:60–30:70, fraction 6 by elution at a gradient of 20:80–10:90, and fraction 7 by elution at a gradient of 0:

100. Fraction 4 was first subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate in a ratio of 8:1–3:1, eluting for 4–5 column volumes. After concentration under reduced pressure, it was then subjected to Sephadex chromatography. LH-20 gel column chromatography was performed using a mixed solvent of petroleum ether:CHCl3:MeOH = 2:1:1 as the eluent. Elution was carried out for 4–5 column volumes. The eluent was concentrated under reduced pressure and then prepared by high-performance liquid chromatography (HPLC). The chromatographic column was a Waters C18, 9.4 × 250 mm, 7 μm, with a flow rate of 2 mL / min and a mobile phase of MeOH:H2O = 80:

20. Compounds 1 and 2 were obtained. All eluent and mobile phase ratios were volume ratios. The fermentation medium mentioned in step (1) is a solid rice culture medium; The structures of compounds 1 and 2 are as follows: 。 2. Use of compound 2 prepared according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-Candida albicans medicament.

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