A medicinal mangrove-derived fungus with anti-tumor activity and its preparation method

Abstract

The present invention relates to an anti-tumor compound derived from a medicinal mangrove fungus and a preparation method thereof. The anti-tumor compound has the structure shown in compound 1-3: #imgabs0#

Description

Technical Field

[0001] The invention belongs to the field of secondary metabolites of mangrove fungi, and particularly relates to an anti-tumor active compound in a medicinal mangrove-derived fungus and a preparation method thereof. Background Art

[0002] Mangroves grow in tropical and subtropical intertidal zones. Their living environment is characterized by high pressure, high salt concentrations, and low oxygen levels. This allows their endophytic fungi to possess unique metabolic pathways, enabling them to produce compounds with novel structures and diverse bioactivities. These metabolites possess diverse medicinal properties, including antibacterial, antitumor, immunomodulatory, and enzyme inhibitory properties. Mangrove endophytic fungi are therefore a potential resource for the development of microbial drugs. Therefore, they are poised to become an important resource for new drug research and development. The applicant's previous Chinese invention patent (CN 116287048 A) disclosed the isolation of a series of diphenyl ether compounds from the fermentation product of the fungus TGGP35. This present invention further investigates TGGP35 and obtains a series of secondary metabolites with antitumor activity. Summary of the Invention

[0003] The present invention provides a medicinal mangrove-derived fungus anti-tumor compound or a pharmaceutically acceptable salt thereof, characterized in that the anti-tumor compound has the structure shown in compound 1-3:

[0004]

[0005] The present invention provides a method for preparing compound 1-3, characterized by comprising the following steps:

[0006] (1) The fungus TGGP35 strain was inoculated into a fermentation medium and cultured at room temperature for 28 to 30 days to obtain a fermentation product;

[0007] (2) extracting the fermented product obtained in step (1) 2 to 4 times with 1 to 2 volumes of ethyl acetate, combining the ethyl acetate phases and concentrating under reduced pressure to obtain an extract;

[0008] (3) The extract obtained in step (2) was subjected to silica gel column chromatography under reduced pressure, using petroleum ether-ethyl acetate as the eluent for gradient elution, the elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and two column volumes were collected for each gradient. The eluate obtained with the gradient of 60:40 was concentrated and then subjected to normal phase silica gel column chromatography, the eluent was a mixed solvent of petroleum ether:ethyl acetate = 10:1-5:1, eluted for 6 column volumes, concentrated under reduced pressure, and then prepared by high performance liquid chromatography HPLC, the chromatographic column was Waters C18, 9.4×250 mm, 7 μm, the flow rate was 2 mL / min, and the mobile phase was MeOH:H2O = 30:70 to 34:66, to obtain compound 1-3; the structure of compound 1-3 is as follows:

[0009]

[0010] The ratio of the eluent or mobile phase is a volume ratio; the fermentation medium is preferably a rice solid medium, and the formula is preferably 50g rice, 60g water, 0.5g sea salt, and 1.5g peptone added to a 1L conical flask.

[0011] The present invention provides an anti-tumor drug, characterized in that any one of the above compounds 1-3 or a pharmaceutically acceptable salt thereof is used as an active ingredient.

[0012] The anti-tumor drug provided by the present invention may also include other anti-tumor drugs and a pharmaceutically acceptable carrier or excipient. The dosage form is preferably a solid preparation or a liquid preparation.

[0013] Another embodiment of the present invention provides the use of any one of the compounds 1-3 or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0014] The term "pharmaceutically acceptable salt" in the present invention refers to non-toxic inorganic or organic acid and / or base addition salts, which can be found in "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201-217.

[0015] The fungus TGGP35 described in this invention was isolated from the medicinal mangrove Acanthus ilicifolius L., collected by the inventors in August 2015 from the Dongzhaigang Mangrove Nature Reserve in the South China Sea. The fungus TGGP35 was identified as Talaromyces flavus (Talaromyces flavus) based on morphological characteristics and molecular biological methods (ITS-rRNA sequence alignment) through 18S rRNA amplification and ITS sequencing. The sequence of the ITS region of this fungus has been submitted to NCBI (GenBank accession No. MT071116). The "fungus TGGP35" described in the present invention has been disclosed in the inventor's previous research paper "Marine Drugs 2022, 20, 361, Talaromarins A–F: Six New Isocoumarins from Mangrove-Derived Fungus Talaromyces flavus TGGP35" and a previous Chinese invention patent (CN116287048 A). The present invention fully cites the contents of the Chinese invention patent (CN 116287048 A). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the strain morphology of TGGP35;

[0017] Figure 2 It is compound 1-3 1 H- 1 H COSY and HMBC (H→C) related signal diagram;

[0018] Figure 3 is the NOESY spectrum of compound 2-3;

[0019] Figure 4 is the experimental ECD spectrum of compound 1;

[0020] Figure 5 is the experimental ECD spectrum of compound 2;

[0021] Figure 6 This is the Mosher's esterification reaction of compound 3 △ δ H Data graph;

[0022] Figure 7 is the 135°-DEPT spectrum of compound 1;

[0023] Figure 8 is the HMQC diagram of compound 1;

[0024] Figure 9 is compound 2 1 H NMR spectrum;

[0025] Figure 10 is compound 2 13 C NMR spectrum;

[0026] Figure 11 is the 135°-DEPT pattern of compound 2;

[0027] Figure 12 is the HMQC diagram of compound 2;

[0028] Figure 13 is the NOESY diagram of compound 2;

[0029] Figure 14 is the 135°-DEPT pattern of compound 3;

[0030] Figure 15 is the HMQC diagram of compound 3;

[0031] Figure 16 is compound 3 1 H- 1 H COSY diagram;

[0032] Figure 17 is the HR-ESI-MS spectrum of compound 3;

[0033] Figure 18 is the HR-ESI-MS spectrum of compound 2. DETAILED DESCRIPTION

[0034] In order to facilitate further understanding of the present invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the invention and are not intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following.

[0035] Example 1

[0036] (1) Prepare the fermentation medium: Add 50 g of rice, 60 g of water, 0.5 g of sea salt, and 1.5 g of peptone to each 1 L Erlenmeyer flask. Sterilize at 120°C for 25–30 minutes.

[0037] The fungus TGGP35 strain was inoculated into a fermentation medium (200 bottles) and cultured at room temperature for 30 days to obtain a fermentation product;

[0038] (2) extracting the fermented product obtained in step (1) three times with equal volumes of ethyl acetate, combining the ethyl acetate phases and concentrating under reduced pressure to obtain an extract;

[0039] (3) The extract obtained in step (2) was subjected to silica gel column chromatography under reduced pressure, and gradient elution was performed using petroleum ether-ethyl acetate as eluent, with the elution gradients being 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. Two column volumes were collected for each gradient, and the eluate obtained with the gradient of 60:40 was concentrated and then subjected to normal phase silica gel column chromatography, with the eluent being a mixed solvent of petroleum ether and ethyl acetate in a ratio of 10:1 to 5:1, eluting for 6 column volumes. After reduced pressure concentration, the extract was subjected to high performance liquid chromatography HPLC, and the chromatographic column was Waters C18, 9.4×250 mm, 7 μm, flow rate 2 mL / min, mobile phase MeOH:H2O=30:70 to 34:66, to obtain compounds 1 (5.3 mg), 2 (5.8 mg), and 3 (6.2 mg); the structures of compounds 1-3 are as follows:

[0040]

[0041] Compound 1: Yellow oil. Combined with the molecular ion characteristics of high-resolution mass spectrometry HR-ESI-MS, a quasi-molecular ion peak [M+H] was given at m / z 225.1473. + , the molecular formula of the compound is deduced to be C 13 H 20 O3, its unsaturation is 4. 1 H-NMR spectrum shows that the compound is in the oxygen-linked region δ H (3.4-5.0) showed two methine hydrogen signals [δ H 4.40 (m, H-5) and 3.47 (m, H-8)], 1 methine δ H 2.48 (m, H-3), in the high field region δ H There are 7 methylene hydrogen signals at (1.5-3.0) [δ H 2.74(m,H-6β),2.53(d,J=3.6Hz,H-2),2.47(m,H-6α),2.38(t,J=7.2Hz,H-11),2.03(m,H -4α),1.74(m,H-4β),1.63(m,H-10α),1.56(m,H-7),1.51(m,H-10β) and 1.38(m,H-9)], in the high field region δ H There is a methyl hydrogen signal at (1.9-2.2) [δ H 1.47(s,H-13)]; combined 13 C-NMR and DEPT135° spectra show that the compound has 13 resonance carbon signals, including 2 keto carbonyl carbon signals δ C(210.2, 209.0), 2 linked oxygenated methine signals δ C (70.0, 67.2), 1 methine signal δ C 27.9, 7 methylene carbon signals δ C (47.2, 47.0, 43.5, 37.5, 36.1, 31.6, 19.6), 1 methyl carbon signal δ C 29.9. According to 1 H- 1 The H COSY spectrum can be used to deduce two related connecting fragments: CH2(6)-CH(5)-CH2(4)-CH(3)-CH2(7) and CH(8)-CH2(9)-CH2(10)-CH2(11). According to the HMBC spectrum, H-6 / H-2 is correlated with C-1, H-2 / 6 / 7 is correlated with C-4, H-2 / 9 is correlated with C-7, H-5 is correlated with C-3, H-11 / 13 is correlated with C-12, and H-13 is correlated with C-11. According to the 1D NOE spectrum, H-3 is correlated with H-5. The planar structure and relative configuration of the compound are determined based on the above spectral data. In addition, the absolute configuration of the compound is determined to be 3S, 5R, 8R by ECD calculation.

[0042] NMR spectrum data of compound 1 (CDCl3)

[0043]

[0044]

[0045] Compound 2: Yellow oil. According to the molecular ion characteristics of high-resolution mass spectrometry HR-ESI-MS, a quasi-molecular ion peak [M+Na] was given at m / z 219.0992. + , the molecular formula of the compound is deduced to be C 11 H 16 O3, its unsaturation is 4. 1 The H-NMR spectrum shows that the compound has three olefin hydrogen signals [δ H 7.05 (s, H-8), 6.81 (m, H-6) and 6.81 (d, J = 16.4 Hz H-6)], in the continuous oxygen zone δ H (5.0-5.2) shows a methine hydrogen signal [δ H 5.03 (d, J = 6.8 Hz, H-9) and 3.84 (td, J = 6.4, 6.0 Hz, H-3)], in the high field region δ H There are two methylene hydrogen signals at (1.5-2.3) [δ H 2.26 (m, H-4) and 1.60 (m, H-3)], in the high field region δH There are two methyl hydrogen signals at (1.2-1.5) [δ H 1.42 (d, J = 6.0 Hz, H-11) and 1.21 (d, J = 6.0 Hz, H-1)]; combined 13 C-NMR and DEPT135° spectra show that the compound has 11 resonance carbon signals, including one ester carbonyl carbon signal δ C 172.1, 4 olefin carbon signals δ C (147.4, 138.1, 129.4, 118.9), 2 oxymethyl signals δ C (77.4, 67.6), 2 methylene signals δ C (38.2, 29.8), 2 methyl carbon signals δ C (23.7,19.3). 1 H- 1 The H COSY spectrum can be used to deduce two related connecting fragments, CH3(1)-CH(2)-CH2(3)-CH2(4)-CH(5)-CH(6) and CH(9)-CH3(11). According to the HMBC spectrum, H-5 is correlated with C-7, H-8 is correlated with C-6, H-9 is correlated with C-7 / 8 / 10, and H-9 is correlated with C-11, respectively. According to the NOESY spectrum, H-6 is correlated with H-8. Based on the above spectral data, the planar structure and relative configuration of the compound were determined. The absolute configuration of C-2 of the compound required Mosher's esterification reaction. Unfortunately, Mosher's esterification reaction was unsuccessful, resulting in the failure to determine the absolute configuration of the hydroxyl group on the side chain C-2 of the compound. The configuration on C-9 was determined to be 9S by ECD calculation.

[0046] NMR spectrum data of compound 2 (CDCl3)

[0047]

[0048]

[0049] Compound 3: Yellow oil. According to the molecular ion characteristics of high-resolution mass spectrometry HR-ESI-MS, a quasi-molecular ion peak [MH] was given at m / z 197.1181. - , the molecular formula of the compound is deduced to be C 11 H 18 O3, its unsaturation is 3. 1 The H-NMR spectrum shows that the compound has three olefin hydrogen signals [δ H6.81 (t, J = 7.2 Hz, H-5), 5.99 (d, J = 11.2 Hz, H-3) and 5.83 (dd, J = 11.2, 6.8 Hz, H-2)], in the continuous oxygen zone δ H (3.5-3.6) shows a methine hydrogen signal δ H 3.53 (m, H-8), in the high field region δ H There are three methylene hydrogen signals at (1.4-2.3) [δ H 2.24 (td, J = 15.4, 7.2 Hz, H-6), 1.61 (m, H-7) and 1.47 (m, H-9)], in the high field region δ H There are two methyl hydrogen signals at (1.0-1.6) [δ H 1.55 (d, J = 6.4 Hz, H-1) and 0.93 (t, J = 7.2 Hz, H-10)]; combined 13 C-NMR and DEPT135° spectra show that the compound has 11 resonance carbon signals, including one ester carbonyl carbon signal δ C 172.4, 4 olefin carbon signals δ C (146.4, 130.4, 128.6, 122.9), 1 oxymethyl signal δ C 72.9, 3 methylene signals δ C (35.4, 30.3, 26.3), 2 methyl carbon signals δ C (15.2,10.0). 1 H- 1 H COSY and HMBC spectra confirmed the connection positions of the structure. Analysis of the NOESY spectrum showed that H-3 was correlated with H-5, confirming that the double bond at C-2 / 4 was cis. The absolute configuration at C-2 of this compound was directly subjected to Mosher's esterification reaction. Based on the chemical shift difference after (S)- and (R)-MTPA esterification reactions, the absolute configuration of C-2 on the side chain was identified as R.

[0050] NMR spectrum data of compound 3 (CDCl3)

[0051]

[0052] Example 2 Cytotoxic activity test:

[0053] 1. Experimental instruments and materials: high-speed centrifuge, clean bench, electronic balance, multifunctional microplate reader, constant temperature incubator, high-pressure sterilizer, DMEM high-glucose culture medium, PBS buffer solution, fetal bovine serum, trypsin, thiazolyl blue, dimethyl sulfoxide (DMSO), adriamycin hydrochloride, centrifuge tubes, cervical cancer cells (HeLa).

[0054] 2. Test method and steps: The cytotoxic activity of monomer compounds 1-3 was tested by MTT method. After the cell line was activated, it was placed in a cell culture incubator (37°C, 5% CO2) for static growth. The cells were used only when the state and number of cells reached the experimental operation level. The cells were digested for 1-3 minutes, and the number of cells was counted with a cell counting plate so that the number of cells in each well of the 96-well plate was 5000. Then a circle of PBS buffer solution was added around it. After the above experimental operation, the plate was placed in a CO2 incubator and stood for 24 hours before adding the test sample. The gradient concentration of the test sample was 20, 10, 5, 2.5, 1.25, and 0.625 μg / mL. The experimental operation was repeated 3 times. After the sample was added, 20 μL MTT was added 48 hours later, followed by 150 μL DMSO. The absorbance value was tested at a wavelength of 492 nm using a microplate reader. DMSO was a negative control and doxorubicin hydrochloride was a positive control. The inhibition rate of each sample was calculated according to the following formula: Inhibition rate (%) = [OD 测试组 -OD DMSO / OD DMSO ] × 100%, and IC was calculated using GraphPad Prism 9 software. 50 value.

[0055] 4. Experimental results:

[0056]

Claims

1. A medicinal anti-tumor compound derived from mangrove fungus or a pharmaceutically acceptable salt thereof, characterized in that The active compound has the structure shown in compound 1-3:

2. A method for preparing compound 1-3, characterized in that The steps include: (1) The fungus TGGP35 strain was inoculated into a fermentation medium and cultured at room temperature for 28 to 30 days to obtain a fermentation product; (2) extracting the fermented product obtained in step (1) 2 to 4 times with 1 to 2 volumes of ethyl acetate, combining the ethyl acetate phases and concentrating under reduced pressure to obtain an extract; (3) The extract obtained in step (2) was subjected to silica gel column chromatography under reduced pressure, using petroleum ether-ethyl acetate as the eluent for gradient elution, the elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and two column volumes were collected for each gradient. The eluate obtained with the gradient of 60:40 was concentrated and then subjected to normal phase silica gel column chromatography, the eluent was a mixed solvent of petroleum ether:ethyl acetate = 10:1-5:1, eluted for 6 column volumes, concentrated under reduced pressure, and then prepared by high performance liquid chromatography HPLC, the chromatographic column was Waters C18, 9.4×250 mm, 7 μm, the flow rate was 2 mL / min, and the mobile phase was MeOH:H2O = 30:70 to 34:66, to obtain compound 1-3; the structure of compound 1-3 is as follows: The fermentation medium in step (1) is selected from rice solid culture medium.

3. An anti-tumor drug, characterized in that Any one of the compounds 1-3 according to claim 1 or a pharmaceutically acceptable salt thereof is used as an active ingredient.

4. The antitumor drug according to claim 3, characterized in that The anti-tumor drug also includes other anti-tumor drugs.

5. The antitumor drug according to any one of claims 3 to 4, characterized in that The anti-tumor drug may further comprise a pharmaceutically acceptable carrier or excipient.

6. The antitumor drug according to claim 5, characterized in that The dosage form of the anti-tumor drug is selected from solid preparations or liquid preparations.

7. Use of any one of compounds 1-3 according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

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