An antioxidant compound from medicinal mangrove fungi and its preparation method

Abstract

This invention relates to an antioxidant-active compound from medicinal mangrove fungi and its preparation method, wherein the antioxidant-active compound has the structures shown in compounds 1-3:

Description

Technical Field

[0001] This invention belongs to the field of secondary metabolites of mangrove fungi, specifically relating to an antioxidant active compound from medicinal mangrove fungi and its preparation method. 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 fosters unique metabolic pathways in their endophytic fungi, enabling them to produce compounds with novel structures and diverse biological activities. These metabolites possess various medicinal values, including antibacterial, antitumor, immunomodulatory, and enzyme-inhibiting properties, making them a potential resource for microbial drug development. Therefore, mangrove endophytic fungi will become an important resource for new drug research. 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 invention further investigates the fungus TGGP35 and obtains an antioxidant-active secondary metabolite. Summary of the Invention

[0003] This invention provides an antioxidant-active compound or a pharmaceutically acceptable salt thereof from medicinal mangrove fungi, characterized in that the antioxidant-active compound has the structures shown in compounds 1-3:

[0004]

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

[0006] (1) Inoculate the fungal strain TGGP35 into the fermentation medium and culture it at room temperature for 28-30 days to obtain the fermentation product.

[0007] (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.

[0008] (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. The eluents obtained from gradients 40:60 and 30:70 were combined and concentrated. Then, the eluents were subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate in the range of 4:1 to 2:1. Eight column volumes were eluted, 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, the flow rate was 2 mL / min, and the mobile phase was MeOH:H2O = 25:75. Compounds 1-3 were obtained. The structures of compounds 1-3 are as follows:

[0009]

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

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

[0012] The antioxidant compositions provided by this invention also contain other antioxidant active ingredients; they may also contain pharmaceutically acceptable carriers or excipients. The preferred dosage form is a solid or liquid formulation.

[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 antioxidant medicament.

[0014] 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.

[0015] The fungus TGGP35 described in this invention was isolated from the medicinal mangrove plant *Acanthus ilicifolius* L., which was collected by the inventors in August 2015 from the Dongzhaigang Mangrove Nature Reserve in the South China Sea, Hainan Province. Fungus TGGP35 was identified as *Talaromyces flavus* (genus *Talaromyces*) based on morphological characteristics and molecular biological methods (ITS-rRNA sequence alignment) using 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 this 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 the previous Chinese invention patent (CN116287048 A). This invention cites in its entirety the contents of the Chinese invention patent (CN 116287048 A). Attached Figure Description

[0016] Figure 1 This is a morphological diagram of the TGGP35 strain;

[0017] Figure 2 It is compounds 1-3 1 H- 1 H COSY Related signal diagram of HMBC (H→C);

[0018] Figure 3 These are the NOESY spectra of compounds 1-3;

[0019] Figure 4 The CD spectra of compounds 1-3 and compounds 1-3 with Mo2 are shown. 4+ Experimental ECD spectrum after complexation;

[0020] Figure 5 These are the experimental ECD spectra of compounds 1-2;

[0021] Figure 6 This is the 135°-DEPT spectrum of compound 1;

[0022] Figure 7 This is the HMBC diagram of compound 1;

[0023] Figure 8 This is the HR-ESI-MS chromatogram of compound 1;

[0024] Figure 9 This is the HMQC diagram of compound 3;

[0025] Figure 10 It is compound 3. 1 H- 1 H COSY diagram;

[0026] Figure 11 This is the HMBC diagram of compound 3;

[0027] Figure 12 This is the NOESY diagram of compound 3;

[0028] Figure 13 This is the HR-ESI-MS image of compound 3. Detailed Implementation

[0029] 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.

[0030] Example 1

[0031] (1) Preparation of fermentation medium: The formula is to add 50g rice, 60g water, 0.5g sea salt and 1.5g peptone to each conical flask (1L conical flask). Incubate at 120℃ for 25–30 minutes.

[0032] The fungal strain TGGP35 was inoculated into fermentation medium (200 bottles) and cultured statically at room temperature for 30 days to obtain the fermentation product.

[0033] (2) The fermentation product obtained in step (1) was extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to obtain an extract.

[0034] (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. The eluents obtained from gradients 40:60 and 30:70 were combined and concentrated. Then, the eluents were subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate in a ratio of 4:1 to 2:1 for 8 column volumes. After vacuum concentration, the eluents were then prepared by high-performance liquid chromatography (HPLC) using a Waters column. C18, 9.4 × 250 mm, 7 μm, flow rate 2 mL / min, mobile phase MeOH:H2O = 25:75, yielded compounds 1 (14.3 mg), 2 (10.4 mg), and 3 (12.1 mg); the structures of compounds 1-3 are as follows:

[0035]

[0036] Compound 1: A yellow oily substance. High-resolution mass spectrometry (HR-ESI-MS) indicates a quasi-molecular ion peak [M+Na] at m / z 235.0939. + Based on the proton and carbon spectral data, the molecular formula of this compound is deduced to be C64. 11 H 16 O4 has an unsaturation degree of 4. 1 The H-NMR spectrum indicates the presence of two active hydrogen signals in this compound [δ]. H 5.06 (s, 9-OH) and 3.59 (s, 10-OH)], three olefin proton signals [δ H 6.98 (dd, J = 12.0, 2.0 Hz, H-6), 6.53 (ddd, J = 15.2, 12.0, 2.0 Hz, H-7) and 6.37 (dd, J = 15.2, 4.8 Hz, H-8)], two methine hydrogen signals appear in the oxygen-bound region [δ H 4.03 (t, J = 4.8 Hz, H⁻⁹) and 3.59 (m, H⁻¹⁰)], 1 methylene hydrogen signal [δ H 4.40 (dd, J = 8.8, 2.4 Hz, H-3α) and 3.59 (dd, J = 8.8, 7.6 Hz, H-3β)] and 1 methylene hydrogen signal δ H At 3.35 m, two methyl signals [δ] appeared in the high-field region. H 1.18 (d, J = 6.8 Hz, H⁻¹²) and δ H 0.97 (d, J = 7.2 Hz, H-11)]; combined with 13C-NMR and DEPT 135° spectra revealed 11 resonance carbon signals in this compound, including one ester carbonyl carbon signal δ. C 171.2, 4 olefin carbon signals δ C (145.6, 134.9, 129.7, 124.5), two δ-hydroxymethyl carbon signals C (74.6, 69.2), 1 hydroxymethylene carbon signal δ C 72.4, δc of 1 methylene hydrogen signal; 31.5, δc of 2 methyl carbon signals. C (20.0, 18.2). Based on the mass spectrometry data, it can be deduced that this compound has 4 degrees of unsaturation. The above proton and carbon spectra only show 3 degrees of unsaturation. The remaining unsaturation needs to form a ring to meet the required number of unsaturation degrees. 1 H- 1 The 1H COSY spectrum shows three linker segments: CH2(3)-CH(4)-CH3(12), CH(6)-CH(7), and CH(8)-CH(9)-CH(10)-CH3(11). Combined with the HMBC spectrum, H-12 is correlated with C-3 / C-5, H-3 / H-6 with C-1, H-6 with C-4, H-8 with C-6, H-9 with C-5, and H-11 with C-10 / C-9. Furthermore, the NOESY spectrum shows a correlation between H-6 and H-8, thus determining the double bond configurations as 6Z and 8E, respectively. Based on the above 1D / 2D NMR data, the planar configuration of this compound is determined. According to the CD test data, a negative Cotton effect is observed at 281 nm, which is consistent with the CD data in the literature. [3] The absolute configuration at C-9 was determined to be 9S by comparison, while the absolute configuration at C-10 required the determination of the CD spectrum of the complex formed in situ in a DMSO solution containing molybdenum acetate. According to the empirical rule of Snatzke's method, the induced CD (ICD) curve observed near 316 nm (Δε-0.42) exhibits a positive Cotton effect, showing the same sign as the OCCO torsion angle in the favorable conformation and allowing for the partition of the absolute configuration, thus confirming that the absolute configuration at C-10 of compound 1 is 10R. In addition, the absolute configuration at C-4 of the compound was determined to be 4R by ECD calculation.

[0037] Nuclear magnetic resonance (NMR) data (DMSO-d6) of compounds 1 and 2

[0038]

[0039] Compound 2: A yellow oily substance. High-resolution mass spectrometry (HR-ESI-MS) and 1D and 2D NMR data indicate that the molecular formula of this compound is C2. 11 H16 O4, with an unsaturation degree of 4, was compared with the 1D NMR data of compound 1. The carbon spectra showed significant similarity, suggesting it might be an epimer. Further analysis... 1 H- 1 Correlation signals in the H COSY and HMBC spectra revealed a connection pattern identical to that of compound 1. Combined with the NOESY spectrum, correlation signals were observed between H-6 and H-8, further confirming the planar structure and relative configuration are consistent with compound 1. CD test data showed a negative Cotton effect at 281 nm. Based on CD data from the literature, the absolute configuration at C-9 was determined to be 9S. According to the empirical rule of Snatzke's method, the induced CD (ICD) curve observed near 311 nm (Δε+1.57) exhibits a negative Cotton effect; therefore, the absolute configuration at C-10 can be determined to be 10S. The absolute configuration at C-4 was determined to be 4R through ECD calculation.

[0040] Compound 3: A yellow oily substance. Based on the molecular ion characteristics observed by high-resolution mass spectrometry (HR-ESI-MS), a quasi-molecular ion peak [MH] is given at m / z 211.0972. - The molecular formula of the compound was deduced to be C. 11 H 16 O4 has an unsaturation degree of 4. 1 The H-NMR spectrum shows that this compound contains three olefin hydrogen signals [δ]. H 7.15 (d, J = 8.8 Hz, H⁻⁵), 6.27 (m, H⁻⁴), 6.02 (dd, J = 11.2, 2.0 Hz, H⁻³), 6.26 (d, J = 5.2 Hz, H⁻⁷) and 5.75 (m, H⁻²)], showing two methylene hydrogen signals in the oxygen-bound region [δ H 3.97 (dd, J = 5.2, 3.2 Hz, H⁻⁸) and 3.55 (m, H⁻⁹)], in δ H Two methyl hydrogen signals exist at (0.9-1.5) [δ] H 1.47 (d, J = 6.8 Hz, H⁻¹) and δ H 0.95 (d, J = 6.4 Hz, H-10)]; combined with 13 C-NMR and DEPT 135° spectra revealed that this compound possesses 11 resonance carbon signals, including one carboxyl carbon signal δ. C 168.3, 6 olefin carbon signals δ C (143.1, 138.2, 128.6, 127.3, 126.5, 124.2), two hydroxymethyl carbon signals δ C (74.7, 69.4), 2 methyl carbon signals δC (18.2, 14.9). According to 1 H- 1 The H COSY spectrum yields two related linking fragments: CH3(10)-CH(9)-CH(8)-CH(7) and CH(5)-CH(4)-CH(3)-CH(2)-CH3(1). According to the HMBC spectrum, H-10 is related to C-8, H-5 to C-1 / 3, and H-8 to C-6. Combined with the NOESY spectrum, we can see that H-7 is related to H-5 and H-4 is related to H-2, further confirming that the hydrogen orientation of the three is the same and the double bond configuration is cis. Based on the above 1D / 2DNMR NMR data, the planar structure and relative configuration of the compound are determined. According to the CD data, a positive Cotton effect exists at 279 nm. Comparison with literature data confirms that the absolute configuration of C-8 is 8R. Furthermore, based on the empirical rule proposed by the Snatzke method, a positive Cotton effect exists at 316 nm (Δε+0.69), thus confirming that the absolute configuration of the compound is 8R,9S.

[0041] Nuclear magnetic resonance (NMR) data of compound 3 (DMSO-d6)

[0042]

[0043] Example 2 Antioxidant Activity

[0044] 1. Testing instruments and materials: Total antioxidant capacity assay kit, PBS buffer solution, 96-well plate, microplate reader, EP tubes and pipettes, etc.

[0045] 2. Dilute the working stock solution from step (1) with PBS buffer to prepare ABTS working solution (35-50 times dilution), and then use a microplate reader to measure its absorbance at 734 and 405 nm as follows:

[0046] A ABTS工作液 -A PBS =A 734 (0.7±0.05); A ABTS工作液 -A PBS =A 405 (around 1.4)

[0047] 3. Antioxidant capacity test:

[0048] Antioxidant activity of compounds 1-3 was tested using a total antioxidant capacity assay kit with PBS as the buffer solution. Sample concentrations were prepared at five gradients: 1.0, 0.75, 0.5, 0.25, and 0.125 mg / mL. For each test, 200 μL of ABTS working solution, 10 μL of the test sample (experimental group), 10 μL of PBS (blank group), and 10 μL of Ltrox (positive control group) were added to a 96-well plate. The test was performed in triplicate. After standing for 5-7 minutes, the absorbance was measured at 734 or 405 nm. The inhibition rate of the test sample was calculated using the following formula: Inhibition rate (%) = [(A...] blank -A sample ) / A blank [×100%, IC calculated using GraphPad Prism 9 software] 50 value.

[0049] 4. Experimental Results:

[0050]

Claims

1. An antioxidant active compound from a mangrove-derived fungus or a pharmaceutically acceptable salt thereof, characterized in that The antioxidant active compound has the structure of compound 3:

2. A process for the preparation of the compound of claim 1, 3, characterized in that The method comprises the following steps: (1) inoculate the fungal TGGP35 strain into a fermentation medium, and incubate at room temperature for 28-30 days to obtain a fermentation product; (2) extract the fermentation product obtained in step (1) with 1-2 volumes of ethyl acetate for 2-4 times, combine the ethyl acetate phases, and concentrate under reduced pressure to obtain an extract; (3) subject the extract obtained in step (2) to reduced-pressure silica gel column chromatography, and perform gradient elution using petroleum ether-ethyl acetate as the eluent, with the elution gradient 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, and collecting two column volumes for each gradient; combine the eluents obtained in the gradients 40:60 and 30:70, concentrate, and then perform normal-phase silica gel column chromatography, with the eluent being a mixture of petroleum ether and ethyl acetate in a ratio of 4:1-2:1, and eluting 8 column volumes; concentrate under reduced pressure, and then perform high-performance liquid chromatography (HPLC) preparation, with the chromatographic column being a Waters C18 column with a size of 9.4*250 mm and a particle size of 7 μm, the flow rate being 2 mL / min, and the mobile phase being MeOH:H2O=25:75, to obtain compound 3; The fermentation medium is a rice solid medium.

3. An antioxidant composition characterized in that The composition comprises compound 3 or a pharmaceutically acceptable salt thereof as an effective ingredient.

4. The composition of claim 3, characterized in that The composition further comprises other antioxidant active ingredients.

5. The composition of any one of claims 3-4, characterized in that The composition further comprises a pharmaceutically acceptable carrier or excipient.

6. The composition of claim 5, wherein The dosage form of the composition is selected from solid preparations or liquid preparations.

7. Use of compound 3 or a pharmaceutically acceptable salt thereof in the preparation of an antioxidant active drug.

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