A marine Aspergillus fungus, xanthones, its preparation method and application

Abstract

This invention belongs to the field of biotechnology and pharmaceutical technology, specifically relating to a marine Aspergillus fungus, a xanthonone compound, its preparation method, and its application. The xanthonone compound is obtained from the marine Aspergillus fungus *Aspergillus versicolor* MSHOU1538 through fermentation, crude extraction, and purification. The selected *Aspergillus versicolor* MSHOU1538 is easy to preserve and cultivate, and the fermentation method facilitates large-scale production. The xanthonone compound obtained from the fermentation process has a novel structure and exhibits Na+ resistance. + / K + -ATPase and Ca 2+ / Mg 2+ -ATPase has an inhibitory effect and its extraction and separation methods are simple, making it a promising candidate drug for antiarrhythmic drugs and creating favorable conditions for the treatment of the disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and medicine, specifically relating to a xanthonone compound prepared using the marine Aspergillus fungus MSHOU1538, its preparation method, and its application. Background Technology

[0002] The marine environment, characterized by high salinity, low temperature, high pressure, and complex light variations, has enabled marine microorganisms to develop unique metabolic mechanisms under these special ecological conditions, producing novel and bioactive secondary metabolites. These secondary metabolites from marine microorganisms are abundant and represent an important source for the discovery of novel bioactive compounds.

[0003] Ion channels are essential components for maintaining cellular ion transport and homeostasis. Among them, Na+... + / K + -ATPases play an important role in maintaining the intracellular and extracellular sodium and potassium ion gradients and cell membrane potential; Ca 2+ / Mg 2+ -ATPases play an important role in regulating intracellular calcium ion concentration and maintaining normal physiological functions. + / K + -ATPase and Ca 2+ / Mg 2+ Abnormal ATPase function is closely related to a variety of cardiovascular diseases, nervous system diseases and metabolic diseases. Therefore, it is of great value to develop active compounds that inhibit or regulate the above-mentioned ATPases.

[0004] In the existing technology, for Na + / K + -ATPase and Ca 2+ / Mg 2+ The regulation of ATPase activity still faces challenges such as limited variety, insufficient selectivity, significant toxicity, and unsatisfactory application effects. Although marine fungal metabolites have shown promising application potential in various bioactivity studies, research on the effects of marine microbial metabolites, especially marine fungal metabolites, on Na+ remains limited. + / K + -ATPase and Ca 2+ / Mg 2+ Research on the inhibitory effects of ATPase is still limited, especially on compounds that simultaneously exhibit the inhibitory effects of the two ATPases mentioned above and their preparation methods.

[0005] Therefore, it is necessary to provide a solution derived from microbial metabolites that is effective against Na+. + / K + -ATPase and Ca 2+ / Mg 2+Compounds that inhibit ATPase and their preparation methods are of significant research importance and application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a xanthonone compound, its preparation method, and its application. This compound is prepared by fermentation culture using the marine Aspergillus fungus MSHOU1538 and can inhibit Na+ in HEK293 cells. + / K + -ATPase and Ca 2+ / Mg 2+ -ATPase activity.

[0007] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a marine Aspergillus fungus, the strain name being Aspergillus versicolor MSHOU1538, classified as Aspergillus versicolor, deposited at the China Center for Type Culture Collection on May 9, 2026, with accession number CCTCC NO: M 2026915.

[0009] Secondly, the present invention provides a method for preparing xanthonesone compounds using the above-mentioned marine Aspergillus fungus MSHOU1538, comprising the following steps:

[0010] (1) Fermentation culture: Aspergillus versicolor MSHOU1538 was inoculated into a fermentation medium containing the following components: sucrose, sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride, Saccharomyces cerevisiae extract, cobalt chloride, ferrous sulfate, calcium chloride, and L-ornithine hydrochloride.

[0011] (2) Crude extraction: The fermented cells were thoroughly mixed in methanol and extracted with ultrasonic assistance. After extraction, the extract was concentrated. The extract was then extracted with chloroform, and the methanol layer was retained. The extract was then extracted with ethyl acetate. The crude extract was obtained by concentrating the ethyl acetate after extraction.

[0012] (3) Separation and purification: The crude extract containing the compound was separated by semi-preparative high performance liquid chromatography. A C18 reversed-phase column was used as the stationary phase, and the organic phase and acidic aqueous solution were used as the mobile phase for gradient elution. The target fraction was collected with a threshold of 20 mV as the automatic receiving signal, thereby obtaining the purified target compound.

[0013] Preferably, in step (1), the mass percentages of each component in the fermentation medium are as follows: sucrose 0.05%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.005%, potassium chloride 0.005%, Saccharomyces cerevisiae extract 0.1%, cobalt chloride 0.25‰, ferrous sulfate 1.5‰, calcium chloride 0.65‰, and L-ornithine hydrochloride 1%; the pH of the fermentation medium is adjusted to 5.8.

[0014] Preferably, in step (1), the fermentation culture conditions are: 28 ℃, 180 r / min shaker culture for 5 days.

[0015] Preferably, in step (2), when using ethyl acetate for extraction, the pH of the methanol layer is first adjusted to 3 using a 1% phosphoric acid aqueous solution.

[0016] Preferably, in step (3), the C18 reversed-phase chromatographic column has a specification of 150 mm × 21.2 mm and a particle size of 5 μm.

[0017] Preferably, in step (3), the organic phase is acetonitrile, and the acidic aqueous solution is a 0.1% hydrochloric acid aqueous solution.

[0018] Preferably, in step (3), the gradient elution program is as follows: at 0 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 90:10; at 14.00 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 43:57; at 14.01 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 5:95; at 17.00 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 5:95; at 17.01 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 90:10; at 20.00 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 90:10; the flow rate is 21.0 mL / min, and the injection volume is 1 mL.

[0019] Thirdly, the present invention provides a xanthonone compound prepared by the above-described preparation method, having the molecular formula Cx. 18 H 12 O7 has the following chemical structural formula (Ⅰ):

[0020]

[0021] (I).

[0022] Furthermore, the structural identification evidence for the xanthones is as follows:

[0023] According to the compound 1 H NMR and 13The C NMR data are in good agreement, indicating that the compound has a highly oxidized fused aromatic heterocyclic skeleton. 13 The presence of two distinct low-field carbon signals at δC 180.75 and 175.04 in the 12C NMR spectrum suggests the presence of a conjugated carbonyl group or a highly deshielded carbonyl structure in the molecule. Meanwhile, multiple low-field aromatic carbon signals appear in the range of δC 154.54–164.90, indicating that multiple oxygen-containing substituents, such as phenolic hydroxyl, methoxy, or ether bonds, are attached to the aromatic ring. 1 In H NMR, the low-field singlet at δH 12.92 can be attributed to the phenolic hydroxyl proton. Its significant low-field shift suggests that the hydroxyl group may form an intramolecular hydrogen bond with a neighboring carbonyl group or oxygen-containing group, further supporting the presence of a conjugated aromatic carbonyl structure with phenolic hydroxyl substitution in the molecule.

[0024] In the aromatic proton region, signals such as δH 7.33, 6.78, 6.36, and 6.19 are related to... 13 The corresponding signals of hydrogen-containing aromatic carbons near δC 115.59 and 130.68 in C NMR indicate the presence of multiple aromatic ring protons in the molecule. The relatively small number of aromatic hydrogens and the abundance of low-field oxygen-substituted aromatic carbon signals suggest a high degree of substitution in the aromatic skeleton, consistent with the characteristics of highly oxidized fused aromatic compounds. Furthermore, the proton signal at δH 8.29 corresponds to the carbon signal near δC 87.96, suggesting that this position may be located in an oxygen-containing fused heterocycle or a special conjugated environment, undergoing deshielding due to the anisotropic effects of oxygen atoms and aromatic rings.

[0025] In the aliphatic region, the proton signals at δH 2.63 and 2.14 correspond to δC 37.45, which can be attributed to the methylene structure in the ring system, indicating the presence of a partially saturated cyclic segment in this compound. The singlet at δH 3.83 corresponds to δC 53.61, consistent with the NMR characteristics of methoxy groups, indicating the presence of methoxy substitution in the molecule. In summary, this compound... 1 H NMR and 13 CNMR data together show that its structure contains conjugated carbonyl groups, phenolic hydroxyl groups, methoxy groups, multiple oxygen-substituted aromatic carbons, aromatic protons, and a partially saturated oxygen-containing fused ring structure, which is consistent with the NMR characteristics of highly oxidized xanthones or related fused aromatic heterocyclic compounds.

[0026] Fourthly, the present invention provides the application of the above-mentioned xanthone compounds, which can inhibit Na+ in HEK293 cells. + / K + -ATPase and Ca 2+ / Mg 2+-ATPase activity holds promise for the development of antiarrhythmic drugs. Specifically, the application is selected from one or more of the following:

[0027] (i) In suppressing Na + / K + -ATPase and Ca 2+ / Mg 2+ -Applications in ATPase activity;

[0028] (ii) Application in the preparation of antiarrhythmic drugs.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The selected Aspergillus versicolor MSHOU1538 fungus is easy to preserve and cultivate, and the fermentation culture method is easy to scale up for production. The xanthones obtained from the fermentation of this strain have novel structures and are effective against Na+. + / K + -ATPase and Ca 2+ / Mg 2+ -ATPase has an inhibitory effect and its extraction and separation methods are simple, making it a promising candidate drug for antiarrhythmic drugs and creating favorable conditions for the treatment of the disease. Attached Figure Description

[0031] Figure 1 Phylogenetic tree diagram of fermentation strains based on ITS.

[0032] Figure 2 Morphological image of the strain after 7 days of culture on a PDA plate.

[0033] Figure 3 Scanning electron microscope (SEM) images of the strain after 7 days of culture on PDA plates. In the figures, (a)-(c) are hyphal structures, and (d)-(f) are spore structures.

[0034] Figure 4 : Liquid chromatogram of crude extract from bacterial fermentation.

[0035] Figure 5 The liquid chromatogram of compound (Ⅰ) after separation by preparative liquid chromatography of crude extract.

[0036] Figure 6 TIC spectrum of compound (Ⅰ).

[0037] Figure 7 : First-order mass spectrum of compound (I).

[0038] Figure 8 Compound of formula (Ⅰ) 1 HNMR spectrum.

[0039] Figure 9 Compound of formula (Ⅰ) 13 CNMR spectrum.

[0040] Figure 10 Compound (I) on the Na+ of HEK293 cells + / K + -ATPase inhibition effect. In the figure, (a)-(c) show the Na2ATPase inhibition effect of HEK293 cells after 24 hours, 48 ​​hours, and 72 hours of culture, respectively. + / K + - ATPase activity detection results; HD, MD, and LD represent the high, medium, and low concentration treatments of the positive drug (Digoxin) control group, respectively; HS, MS, and LS represent the high, medium, and low concentration treatments of xanthones, respectively.

[0041] Figure 11 Compound (I) on Ca2+ in HEK293 cells 2+ / Mg 2+ -ATPase inhibition effect. In the figure, (a)-(c) show the Ca2+ inhibition effect of HEK293 cells after 24 hours, 48 ​​hours, and 72 hours of culture, respectively. 2+ / Mg 2+ - ATPase activity detection results; HV, MV, and LV represent the high, medium, and low concentration treatments of the positive control group (Verapamil), respectively; HS, MS, and LS represent the high, medium, and low concentration treatments of xanthones, respectively. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.

[0043] Example 1: Screening and Identification of Strains

[0044] This embodiment provides the screening and identification process for the marine Aspergillus versicolor MSHOU1538 fungus, as detailed below:

[0045] I. Isolation and Screening of Strains

[0046] Marine mud samples were collected from Zhoushan, Zhejiang Province, and sealed in centrifuge tubes for preservation. 5.0 g of the marine mud sample was added to an Erlenmeyer flask containing 45 mL of sterile physiological saline and shaken for 30 min to ensure thorough dispersion of microorganisms. After standing for 5 min, the supernatant was collected and serially diluted 10-fold with sterile physiological saline to obtain 10... -1 ~10 -5 Serial dilutions. Spread 100 μL of each serial dilution evenly onto potato dextrose agar plates. Incubate the plates upside down at 28°C for 14 days, observing colony growth periodically. Select single colonies showing differences in color, morphology, edge, surface texture, or growth rate, and transfer them to fresh potato dextrose agar for purification using the streak plating method. Repeat purification until a pure culture with consistent colony morphology and uniform microstructure is obtained. Prepare a bacterial suspension containing 20% ​​glycerol from the purified strain and store at -80°C.

[0047] The MSHOU1538 strain was obtained through the above screening process.

[0048] II. Identification of the strain

[0049] The selected MSHOU1538 strain was first activated by culturing it on PDA medium (20% potato juice, 2% glucose, 2% agar) for 5 days, and then the following experiments were performed:

[0050] (1) Morphological observation: A coverslip was placed at a certain angle on the surface of the PDA solid medium used to culture the marine microorganism MSHOU1538, allowing the fungus to naturally crawl onto the coverslip. After culture, the coverslip covering the marine microorganism MSHOU1538 was removed and immersed in 2.5% glyceraldehyde fixation solution. It was fixed at 4°C for 2 hours. It was rinsed three times with phosphate-buffered saline, 10 min each time; and immersed in 1% stannous oxide solution at 4°C for 2 hours. It was dehydrated successively with 50%, 70%, 95% and 100% ethanol, 10 min each time. Tert-butanol substitution was performed, followed by freeze drying, fixation, gold plating, and then microscopic examination. The microscopic results showed ( Figure 2-3 After culturing on PDA medium for 7 to 10 days, the strain developed a mature sporulation structure, including a spherical ascus with relatively smooth mycelial cords; its nutrient-absorbing hyphae are abundant, mainly slender, sparsely branched, and creeping hyphae; in contrast, the more robust erect hyphae undertake the reproductive function; the apex of the sporogen has spores with a decorative surface composed of fine spines or rough textures; these spores are supported by one or two pedicels and arranged in an open radial pattern.

[0051] (2) ITS rDNA sequence alignment: ITS rDNA gene information of the strain was extracted using magnetic beads. The fungal ITS rDNA sequence was amplified using universal fungal primers (ITS1: TCCGTAGGTGAACCTGCGG (SEQ ID NO.1), ITS4: TCCTCGCTTATTGATATGC (SEQ ID NO.2)) as upstream and downstream primer sequences. The ITS rDNA sequence is as follows:

[0052] (SEQ ID NO.3)

[0053] The sequencing results were BLAST aligned and a phylogenetic tree was constructed. The phylogenetic tree of the fermentation strain based on ITS is as follows: Figure 1 As shown, the strain was identified as *Aspergillus versicolor*. The strain was named *Aspergillus versicolor* MSHOU1538 and deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2026, with accession number CCTCC NO: M 2026915.

[0054] Example 2: Preparation of Xanthone Compounds

[0055] (1) Fermentation culture of the strain

[0056] After culturing Aspergillus versicolor MSHOU1538 on PDA medium (20% potato juice, 2% glucose, 2% agar) at 28°C for 5 days, a loopful of bacteria was inoculated into seed culture medium (3.5% glucose, 1% soluble starch, 2% defatted soybean flour, 0.5% bacterial peptone, 0.5% beef extract peptone, 0.3% yeast extract, 0.2% sodium chloride, 0.05% dipotassium hydrogen phosphate, 0.005% magnesium sulfate, pH 5.8) and incubated on a shaker at 28°C. The culture was carried out at a rotation speed of 180 rpm for 3 days. Afterward, a 5% inoculum was added to the fermentation medium (0.05% sucrose, 0.3% sodium nitrate, 0.05% dipotassium hydrogen phosphate, 0.005% magnesium sulfate, 0.005% potassium chloride, 0.1% *Saccharomyces cerevisiae* extract, 0.25‰ cobalt chloride, 1.5‰ ferrous sulfate, 0.65‰ calcium chloride, 1% L-ornithine hydrochloride, pH 5.8), and cultured on a shaker at 28℃ and a rotation speed of 180 rpm for 5 days. The fermentation broth of this strain was obtained.

[0057] (2) Crude extraction of product

[0058] The fermentation broth of the strain was centrifuged (8000 r / min, 15 min), and the supernatant was discarded to obtain bacterial cells. The bacterial cells were thoroughly mixed with anhydrous methanol at a mass-to-volume ratio of 1:1, and extracted three times with ultrasonic assistance, each time for 10 min, followed by centrifugation (8000 r / min, 15 min) to collect the supernatant. The mixture was centrifuged again (8000 r / min, 15 min), and the resulting liquid was concentrated by rotary evaporation. Subsequently, it was extracted with chloroform, retaining the methanol phase. The pH of the methanol layer was adjusted to 3 with 1% phosphoric acid aqueous solution, and then extracted three more times with ethyl acetate to fully obtain the product. Residual water was removed with 5% anhydrous sodium sulfate, and the extracted ethyl acetate was concentrated by rotary evaporation to an oily state to obtain the crude extract of metabolites.

[0059] (3) Separation and purification

[0060] The crude extract was separated and purified using semi-preparative high-performance liquid chromatography (HPLC). An Agilent 1290 Infinity II semi-preparative HPLC system was used, with a Sepax HP-C18 column (150 mm × 21.2 mm, 5 μm). Gradient elution was employed, with acetonitrile and 0.1% (v / v) hydrochloric acid aqueous solution as the mobile phase. The gradient program was: 0 min, acetonitrile / 0.1% hydrochloric acid aqueous solution = 90 / 10 (v / v); 14.00 min, 43 / 57; 14.01 min, 5 / 95; 17.00 min, 5 / 95; 17.01 min, 90 / 10; 20.00 min, 90 / 10; the flow rate was 21.0 mL / min, and the injection volume was 1 mL. During separation, an automatic detector and fraction collector were connected for collection. A threshold of 20 mV was used as the automatic receiving signal, and target fractions exceeding the threshold (≥20 mV) were collected.

[0061] Example 3: Structural characterization of xanthones

[0062] I. HPLC Purity and Fingerprint Analysis

[0063] HPLC analysis was performed using a Waters 1525 high-performance liquid chromatography system with an XTRA MSC18 column (250 mm × 4.6 mm, 5 mm). The system was run at a gradient program at 1 mL / min, using acetonitrile (A) and water (B) as solvents. The solvent gradient combinations were as follows: 0–10 min 30% B; 10–20 min 30–60% B; 20–25 min 60–90% B; 25–30 min 90–60% B. The eluent was monitored at 254 nm.

[0064] The liquid chromatogram of the crude extract in Example 2 is as follows: Figure 4 As shown, the compound can be well separated under these elution conditions and eluted with approximately 50% organic phase; the liquid chromatogram of the target fraction is shown below. Figure 5 As shown, the purity of the separated compound is high. The purity is calculated to be 90.47% by dividing the target peak area by the total peak area.

[0065] II. Structural Analysis

[0066] (1) Liquid chromatography-mass spectrometry (LC-MS / MS): Mobile phase conditions for LC-MS / MS analysis: Initial conditions: 10% acetonitrile and 90% water (containing 0.1% TFA) maintained for 0-20 min. A linear gradient transition was applied within 0-20 min to 50% acetonitrile and 50% aqueous phase (containing 0.1% TFA). From 40-60 min, the composition was restored to 10% acetonitrile and 90% aqueous phase (containing 0.1% TFA). The final equilibration step was maintained at 10% acetonitrile for 60.1-65 min. Mass spectrometry analysis was performed using a positive ion source mode, with the nebulizer gas flow rate maintained at 3 L / min and the drying gas flow rate set at 10 L / min. Temperature control was achieved by stabilizing the interface temperature at 300℃ and maintaining the desolventizing temperature at 526℃. Full scan mass spectrometry analysis was performed in the m / z range of 20-1000 to ensure comprehensive detection of analytes. All experimental conditions were strictly controlled to maintain the repeatability of the method and analytical sensitivity. The results showed ( Figure 6-7 The ion chromatogram detected a sharp peak at 21.50 min, indicating the presence of a peak with m / z of 363.10660 ([M+Na)) in positive mode. + ] + Secondary metabolites of ) predominated in the sample. Based on retention time, UV absorption characteristics, and MS / MS fragmentation mode, the main components were inferred to be xanthones.

[0067] (2) Nuclear Magnetic Resonance Spectroscopy: The purified compound was concentrated to dryness under reduced pressure and then freeze-dried. It was then dissolved in deuterated methanol for NMR analysis. NMR spectra were obtained using a Bruker Avance III 400 MHz NMR spectrometer. 1 H and 13 C spectrum. 1 HNMR and 13 C-NMR spectra were obtained at the analysis center of Shanghai Microspectrochemical Technology Service Co., Ltd.

[0068] The results show that ( Figure 8-9 ), the compound 13 The presence of distinct low-field signals at δC 180.75 and 175.04 in C NMR indicates the presence of a conjugated carbonyl structure in the molecule. Multiple carbon signals distributed in the δC range of 154.54–164.90 suggest that the compound contains multiple oxygen-substituted aromatic carbons and exhibits characteristics of a highly oxidized fused aromatic skeleton. 1In the 1H NMR spectrum, δH 12.92 indicates a significant low-field proton signal for the phenolic hydroxyl group, suggesting that the hydroxyl group may undergo a significant low-field shift due to intramolecular hydrogen bonding. In the aromatic proton region, signals such as δH 7.33, 6.78, 6.36, and 6.19 indicate the presence of multiple aromatic ring protons, with some aromatic hydrogens in a highly substituted aromatic ring environment. The signal at δH 8.29, combined with δC 87.96, suggests that this position may be related to an oxygen-fused heterocycle or a special deshielding environment. In the aliphatic region, δH 2.63 and 2.14 correspond to δC 37.45, which can be attributed to methylene signals in the ring system; δH 3.83 corresponds to δC 53.61, consistent with the NMR characteristics of a methoxy group. Overall, the structure contains a conjugated carbonyl group, a phenolic hydroxyl group, a methoxy group, multiple oxygen-substituted aromatic carbons, and an oxygen-fused heterocycle fragment, conforming to the structural characteristics of highly oxidized fused aromatic compounds. Therefore, the structure is deduced as follows (I):

[0069]

[0070] (I).

[0071] Example 4: Activity evaluation of xanthones

[0072] (1) Na + / K + -ATPase activity assay:

[0073] To evaluate the effect of the xanthone compounds prepared in Example 2 on Na + / K + The inhibitory effect of ATPase activity was investigated by adding xanthones to HEK293 cell culture medium. A blank control group, a positive control group (Digoxin), and a xanthones treatment group were established, and cells were cultured for 24 h, 48 h, and 72 h. The blank control group consisted of HEK293 cell culture medium without the drug. The Digoxin control group and the xanthones treatment group were treated with three drug concentrations: high, medium, and low (0.0625 mg / mL, 0.0312 mg / mL, and 0.0156 mg / mL), respectively, using complete HEK293 cell culture medium as the diluent.

[0074] After culture, the Na+ content of HEK293 cells was measured using the ouabain-sensitive ATP hydrolysis method. + / K +-ATPase activity. The specific steps are as follows: Cells treated with the drug were placed on ice and homogenized in pre-cooled 150 mM histidine buffer (pH 7.4). The resulting homogenate was centrifuged at 4 °C and 20,000 × g for 30 min. The supernatant was used as the enzyme source, and the protein concentration was adjusted to 0.5 mg / mL using a protein quantification method. A 1% saponin solution was added to the enzyme source at a volume ratio of 1:4, along with pyrophosphate and glycerophosphate to a final concentration of 2.7 mM. The mixture was incubated at room temperature for 15 min. Each sample was then divided into a group without ouabain and a group containing ouabain. Both reaction systems contained 121.5 mM NaCl, 19.6 mM KCl, 3.92 mM MgCl2, and 2.94 mM ATP at final concentrations. The ouabain-containing system was treated with ouabain to a final concentration of 1.47 mM, while the ouabain-free system was treated with an equal volume of ultrapure water. All components except ATP were premixed, and ATP was added last to initiate the reaction. The mixture was incubated at 37 °C for 30 min. After the reaction, 50% trichloroacetic acid was added at a volume ratio of 1:10 to the reaction solution to terminate the reaction. The mixture was centrifuged at 3,000 × g for 5 min, and the supernatant was collected. The inorganic phosphorus content in the supernatant was determined at 750 nm using the Taussky-Shorr colorimetric method. + / K + -ATPase activity was calculated as the difference in the amount of inorganic phosphorus released between the ouabain-free group and the ouabain-containing group.

[0075] The results show that ( Figure 10 After culturing HEK293 cells for 24, 48, and 72 hours, the Na+ content of the control group was significantly lower. + / K + -ATPase activity increased with prolonged culture time, possibly related to the stable adhesion, gradual maturation of cell state, and enhanced membrane ion transport function of HEK293 cells during culture. Compared with the blank control group, at high concentrations of the positive control drug, the Na+ level in HEK293 cells during the same period was significantly lower. + / K + -ATPase activity decreased significantly, and with decreasing digoxin concentration, the effect on Na+ decreased. + / K + The inhibitory effect of ATPase was significantly weakened in the group treated with xanthones. + / K + -ATPase activity also decreased, following a trend consistent with that of positive digoxin. However, at the same concentration, positive digoxin showed reduced activity against Na+. + / K+ The inhibitory effect of this compound on ATPase activity is stronger than that of xanthone compounds. Although this compound has a stronger inhibitory effect on Na+... + / K + -ATPase has a weaker inhibitory effect than the positive control, but it may have potential advantages in terms of mildness of action, safety, selectivity or functional protection. Therefore, its application value should be evaluated in combination with multidimensional efficacy indicators.

[0076] (2) Ca 2+ / Mg 2+ -ATPase activity assay:

[0077] To evaluate the effect of the xanthone compounds prepared in Example 2 on Ca2+ in the plasma membrane of HEK293 cells. 2+ / Mg 2+ The effect of ATPase activity was investigated, and HEK293 cells were divided into a blank control group (no drug treatment), a positive drug control group, and a xanthondroitin compound treatment group. The positive drug control group (Verapamil) and the xanthondroitin compound treatment groups were each treated with three concentrations: 0.0625 mg / mL, 0.0312 mg / mL, and 0.0156 mg / mL. HEK293 cells in each group were treated for 24, 48, and 72 h, respectively.

[0078] Cells were collected after treatment, and plasma membrane protein components were prepared and protein concentrations were determined. The calcium content of the plasma membrane components was determined using an inorganic phosphorus release assay. 2+ Activated Mg 2+ Dependent ATPase activity. The specific steps are as follows: The total reaction volume is 250 μL. Each reaction system contains 250 μg of plasma membrane protein and a final concentration of 0.25 mM MgATP, 50 mM Tris-HCl buffer (pH 8.0), and 0.4 mM EGTA. Each sample is prepared with Ca... 2+ Missing control group and Ca 2+ The assay group: no additional CaCl2 was added to the control group; the assay group added an appropriate amount of CaCl2 based on the Ca–EGTA equilibrium calculation to increase the free Ca in the system. 2+ The concentration was approximately 0.1 μM. All components except MgATP were mixed and pre-incubated at 30 °C. Then, MgATP was added to initiate the reaction, and the reaction was continued at 30 °C for 10 min. After the reaction, acidic malachite green-ammonium molybdate colorimetric stop solution was added, and the absorbance was measured at 660 nm. The amount of inorganic phosphorus generated was calculated based on the KH₂PO₄ standard curve. Ca 2+ Activated Mg 2+ ATPase-dependent activity with Ca 2+ The test group and Ca 2+The difference in inorganic phosphorus release between the missing control group and the control group was calculated. A reagent blank and a zero time blank without plasma membrane proteins were also set up to correct for background inorganic phosphorus from ATP, reagents, and the sample itself.

[0079] The results showed that ( Figure 11 Xanthone compounds can significantly inhibit Ca2+ in HEK293 cells. 2+ / Mg 2+ The compound exhibited ATPase activity, with its inhibitory effect generally comparable to that of the positive control drug, and even superior to the positive control drug under certain experimental conditions, indicating that this compound possesses strong Ca2+ inhibitory activity. 2+ / Mg 2+ -ATPase inhibitory activity could serve as a potential active candidate.

[0080] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A marine Aspergillus fungus, characterized in that, The strain is named Aspergillus versicolorMSHOU1538, classified as Aspergillus versicolor, and deposited at the China Center for Type Culture Collection (CCTCC) on May 9, 2026, with accession number CCTCC NO: M 2026915.

2. A method for preparing xanthones, characterized in that, The xanthonone compounds are prepared using the marine Aspergillus fungus described in claim 1, comprising the following steps: (1) Fermentation culture: Marine Aspergillus fungi were inoculated into a fermentation medium containing the following components: sucrose, sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride, Saccharomyces cerevisiae extract, cobalt chloride, ferrous sulfate, calcium chloride, and L-ornithine hydrochloride. (2) Crude extraction: The fermented cells were thoroughly mixed in methanol and extracted with ultrasonic assistance. After extraction, the extract was concentrated. The extract was then extracted with chloroform, and the methanol layer was retained. The extract was then extracted with ethyl acetate. The crude extract was obtained by concentrating the ethyl acetate after extraction. (3) Separation and purification: The crude extract containing the compound was separated by semi-preparative high performance liquid chromatography. A C18 reversed-phase column was used as the stationary phase, and the organic phase and acidic aqueous solution were used as the mobile phase for gradient elution. The target fraction was collected with a threshold of 20 mV as the automatic receiving signal, thereby obtaining the purified target compound.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass percentages of each component in the fermentation medium are as follows: sucrose 0.05%, sodium nitrate 0.3%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.005%, potassium chloride 0.005%, Saccharomyces cerevisiae extract 0.1%, cobalt chloride 0.25‰, ferrous sulfate 1.5‰, calcium chloride 0.65‰, and L-ornithine hydrochloride 1%; the pH of the fermentation medium is adjusted to 5.

8.

4. The preparation method according to claim 2, characterized in that, In step (1), the fermentation culture conditions are: 28 ℃, 180 r / min shaker culture for 5 days.

5. The preparation method according to claim 2, characterized in that, In step (2), when using ethyl acetate for extraction, the pH of the methanol layer is first adjusted to 3 using a 1% phosphoric acid aqueous solution.

6. The preparation method according to claim 2, characterized in that, In step (3), the organic phase is acetonitrile, and the acidic aqueous solution is a 0.1% hydrochloric acid aqueous solution.

7. The preparation method according to claim 2, characterized in that, In step (3), the gradient elution program is as follows: at 0 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 90:10; at 14.00 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution is 43:

57. At 14.01 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution was 5:95; at 17.00 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution was 5:95; at 17.01 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution was 90:10; at 20.00 min, the volume ratio of acetonitrile to 0.1% hydrochloric acid aqueous solution was 90:10; the flow rate was 21.0 mL / min, and the injection volume was 1 mL.

8. A xanthonone compound, characterized in that, Its molecular formula is C 18 H 12 O7 has the following chemical structural formula (Ⅰ): ； （Ⅰ）。 9. The xanthonesone compound according to claim 8, characterized in that, It is prepared by the preparation method described in any one of claims 2-7.

10. The use of the xanthone compound prepared by the method according to any one of claims 2-7 or the xanthone compound according to claim 8, selected from one or more of the following: (i) In suppressing Na + / K + -ATPase and Ca 2+ / Mg 2+ -Applications in ATPase activity; (ii) Application in the preparation of antiarrhythmic drugs.

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