A fungus and its method for preparing Demethylincisterol A3 and a drug for acute liver injury

Abstract

The present invention provides a fungus, which is a Cordyceps family fungus ( Parengyodontium album ) SDGP-1; the Cordyceps fungus SDGP-1 is deposited with the China Center for Type Culture Collection with the accession number CCTCC M 2025301. The fungus of the present invention is used to prepare the active natural product demethylincisterol A3, overcoming the drawbacks of demethylincisterol A3 in nature, such as its low abundance, seasonal availability, and low production efficiency, allowing for large-scale production. Furthermore, this compound has promising applications in liver protection.

Description

Technical Field

[0001] The present invention relates to a kind of fungus Parengyodontium album , in particular to a fungus whose fermented mycelium contains the compound Demethylincisterol A3 Parengyodontium album , as well as the cultivation method of this fungus and the preparation method of the secondary metabolite Demethylincisterol A3 and its application in liver protection, belonging to the field of microbial technology. Background Art

[0002] The compound Demethylincisterol A3 is a kind of demosterol compound. Research shows that this compound has significant inhibitory activities against various cancer cell lines such as human breast cancer cell line MCF-7 and colon cancer cell line HT-29, and bacteria such as Staphylococcus aureus, Escherichia coli, and Helicobacter pylori, and can also have a good lethal effect on brine shrimp. In recent years, researchers have isolated this compound from the secondary metabolites of various fungi, but the content is extremely low (<0.1%), making it impossible to achieve large-scale preparation of this compound. The wild edible fungus Gyroporus purpureus Gomphus purpuraceus (Iwade) K. Yokoy.] fruit bodies contain this compound, with a content of 0.001%, and it is restricted by seasonal production. The present invention isolates and obtains a strain of fungus ( Parengyodontium album ) SDGP-1 from the fruit bodies of the wild edible fungus Gyroporus purpureus. The mycelium contains Demethylincisterol A3, and the isolation yield is 4.8%, which is higher than the yield reported in the existing literature, and can be used for large-scale and continuous preparation and production of Demethylincisterol A3, providing an important resource for its application expansion.

[0003]

[0004] Structural formula of the compound Demethylincisterol A3. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of fungus that can produce the active natural product Demethylincisterol A3 through fermentation or prepare an extract containing the natural product Demethylincisterol A3.

[0006] The present invention provides a strain of fungus SDGP-1( Parengyodontium album ) ; the Cordycipitaceae fungus SDGP-1 is preserved in the China Center for Type Culture Collection, Wuhan, Hubei, with the preservation number CCTCC M 2025301, and the preservation date is February 25, 2025.

[0007] On the one hand, the present invention provides a method for preparing the natural product Demethylincisterol A3, which is extracted and separated from mycelium after fermentation of the said fungus, and includes the following steps:

[0008] (1) Inoculate the said fungus SDGP-1 into a seed medium for cultivation to obtain a seed solution of strain SDGP-1;

[0009] (2) Transfer the seed solution of strain SDGP-1 to a fermentation medium for cultivation to obtain mycelium containing the natural product Demethylincisterol A3;

[0010] (3) The mycelium containing the natural product Demethylincisterol A3 is filtered, and after separating the mycelium, it is dried, extracted, and concentrated under reduced pressure to obtain an extract of Demethylincisterol A3;

[0011] (4) The extract of Demethylincisterol A3 is separated by resin, eluted with an alcohol solution, subjected to reverse-phase column chromatography, eluted with an alcohol solution, purified by high-performance liquid chromatography, and eluted with an acetonitrile solution to obtain the natural product Demethylincisterol A3.

[0012] The formulations of the said seed medium and fermentation medium are the same, and are both products obtained by boiling peeled potatoes, mixing them with glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and peptone, fully dissolving them, and then fixing the volume with water and sterilizing.

[0013] In some preferred cases, first inoculate the fungus SDGP-1 into a sterilized seed medium and cultivate it in a constant temperature shaking incubator at 24°C (150 r / min) for 14 days to obtain a seed solution of strain SDGP-1. Then transfer the seed solution to a sterilized fermentation medium and continue to cultivate it in a constant temperature shaking incubator at 24°C (150 r / min) for 14 days. The formulations of the seed medium and fermentation medium are the same, and are expressed as: cut peeled potatoes into pieces and boil for 30 min, filter its juice, mix it with glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and peptone, fully dissolve them, fix the volume with water, the pH value is natural, and sterilize at 115°C for 30 minutes. In the finally fixed volume of the medium, calculated by a volume of 1 L, the starting material masses are: 200 g of peeled potatoes, 20 g of glucose, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, and 2 g of peptone. For the fermentation medium, the amounts of raw materials can be enlarged in multiples.

[0014] In some preferred cases, after the fungal strain SDGP-1 is fermented and cultured, the isolation and preparation of Demethylincisterol A3 is carried out according to the following procedure: the fermentation broth is filtered to separate the mycelium, which is dried in a constant temperature drying oven and then extracted three times with ethyl acetate. The extract is concentrated under reduced pressure using a rotary evaporator to obtain an extract of Demethylincisterol A3. The extract is separated by macroporous resin chromatography and gradient elution is carried out with an ethanol / water system (60 / 40 - 100 / 0), and fractions are collected in segments to obtain 5 components. The component containing the target chemical is detected using a high performance liquid chromatograph, and all components containing the target compound are combined and subjected to reverse phase column chromatography. Gradient elution is carried out with a methanol / water system (70 / 30 - 100 / 0), and after high performance liquid chromatographic analysis, the components containing the target compound are combined and further purified by semi-preparative high performance liquid chromatography. Isocratic elution is carried out with an acetonitrile / water system (70 / 30) to obtain the target compound Demethylincisterol A3.

[0015] The temperature of the constant temperature drying oven is 45 - 50 °C.

[0016] The macroporous resin is D101. The separation step of the macroporous resin column is gradient elution with an ethanol / water system (60 / 40 - 100 / 0), and the components containing Demethylincisterol A3 are detected using high performance liquid chromatography and combined to obtain the enriched fraction of the macroporous resin column.

[0017] The reverse phase column packing is C18, and the chromatography separation step is gradient elution with a methanol / water system (70 / 30 - 100 / 0), and the components containing Demethylincisterol A3 are detected using high performance liquid chromatography and combined to obtain the enriched fraction of the reverse phase column.

[0018] The detection conditions for the high performance liquid chromatography are as follows: the chromatographic column is a YMC-Pack ODS-A series C18 chromatographic column (4.6 mm × 250 mm, 5 μm), 1‰ acetic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is carried out (0 - 40 min, B / A: 10% → 90%; 40 - 55 min, B / A: 90% → 100%; 55 - 75 min, B / A: 100% → 100%), the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0019] The purification conditions for the semi-preparative high performance liquid chromatography are isocratic elution with an acetonitrile / water system (70 / 30), and the detection wavelength is 217 nm.

[0020] Another aspect of the present invention is to provide a liver-protecting health product, which is characterized by comprising the extract of Demethylincisterol A3 or the natural product Demethylincisterol A3.

[0021] Use of the liver-protecting health product in increasing the expression of antioxidant signaling pathway proteins in hepatocytes AML12, wherein the expression of antioxidant signaling pathway proteins in hepatocytes AML12 includes the increase in the expression of HO-1 protein, p62 and the phosphorylation level of p62 in AML12 cells.

[0022] Use of the liver-protecting health product in increasing the transcriptional expression of antioxidant genes in hepatocytes AML12, wherein the transcriptional expression of antioxidant genes in hepatocytes AML12 includes the increase in the expression of antioxidant protein gene HO-1 and glutamate-cysteine ligase.

[0023] A drug for acute liver injury, comprising the extract of Demethylincisterol A3 or the natural product Demethylincisterol A3.

[0024] Pharmaceutical use of the extract of Demethylincisterol A3 or the natural product Demethylincisterol A3 in increasing the levels of alanine aminotransferase and aspartate aminotransferase in serum in acute liver injury diseases.

[0025] The drug for acute liver injury is used for the treatment of acute liver injury diseases by intraperitoneal injection.

[0026] The present invention isolates a fungus ( Parengyodontium album ) SDGP-1 from the fruiting bodies of the wild edible fungus Gyroporus castaneus. The mycelium contains Demethylincisterol A3, and the isolation yield is 4.8% or more, which can be used for large-scale and continuous preparation and production of Demethylincisterol A3, providing an important resource for its application expansion. Description of the Drawings

[0027] Figure 1 : Liquid chromatography diagram of the ethyl acetate extraction extract of SDGP-1 mycelium.

[0028] Figure 2 : Liquid chromatography diagram of Demethylincisterol A3.

[0029] Figure 3 : Proton nuclear magnetic resonance spectrum for nuclear magnetic identification of the compound Demethylincisterol A3 fermented by SDGP-1.

[0030] 1H NMR analysis conditions: Measured on a Bruker-ARX-400 nuclear magnetic resonance spectrometer, with deuterated chloroform as the solvent and tetramethylsilane as the internal standard.

[0031] Figure 4 : NMR identification of carbon spectrum of Demethylincisterol A3 compound fermented by SDGP-1.

[0032] Figure 5 : Changes in antioxidant proteins after Demethylincisterol A3 at different concentrations acted on mouse hepatocytes (AML12) for 24 h.

[0033] Figure 6 : Gray-scale analysis of antioxidant protein bands after Demethylincisterol A3 at different concentrations acted on mouse hepatocytes (AML12) for 24 h.

[0034] Figure 7 : Changes in antioxidant genes after Demethylincisterol A3 at different concentrations acted on mouse hepatocytes (AML12) for 24 h.

[0035] Figure 8 : Changes in the levels of ALT and AST in the sera of mice in different groups.

[0036] Figure 9 : Staining results of H&E sections of the livers of mice in different groups. Detailed implementation mode

[0037] To elaborate on the technical solutions and technical objectives of the present invention, the present invention will be further introduced below in conjunction with the drawings and specific implementation modes.

[0038] Example 1 Liquid fermentation of SDGP-1

[0039] 1. Preparation of seed liquid:

[0040] Weigh 200 g of peeled potatoes, cut them into pieces and boil for 30 min. After filtering the boiling juice, mix it with 20 g of glucose, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, and 2 g of peptone and dissolve them fully. Add water to make the volume up to 1 L, and the pH value is natural. Aliquot it into 250 mL conical flasks, with 100 mL of liquid medium in each flask. After sealing the conical flasks, place them in a high-pressure steam sterilizer and sterilize at 115 °C for 30 minutes. After cooling, inoculate the fungus SDGP-1 and shake culture it in a constant temperature shaking incubator at 24 °C and 150 r / min for 14 days to obtain the seed liquid of SDGP-1.

[0041] 2. Preparation of fermentation broth:

[0042] Weigh 4 kg of peeled potatoes, cut them into pieces and boil for 30 min. After filtering the boiling juice, mix it with 400 g of glucose, 60 g of potassium dihydrogen phosphate, 30 g of magnesium sulfate heptahydrate, and 40 g of peptone, and fully dissolve them. Then add water to make the volume up to 20 L, and the pH value is natural. Aliquot it into 3-L conical flasks, with 1.5 L of liquid medium in each flask. After sealing the conical flasks, sterilize them in a high-pressure steam sterilizer at 115 °C for 30 minutes. After cooling, inoculate with the SDGP-1 seed liquid and shake culture in a constant temperature shaking incubator at 24 °C and 150 r / min for 14 days to obtain the SDGP-1 fermentation broth.

[0043] Preparation and Identification of Demethylincisterol A3 in the Fermentation Product of Example 2

[0044] 1. Pretreatment of the Fermentation Product

[0045] Filter the SDGP-1 fermentation broth in Example 1 using a 120-mesh nylon mesh cloth to separate the mycelium. After drying the mycelium in a constant temperature drying oven, crush it with a pulverizer, extract it 3 times with ethyl acetate, and concentrate the extract under reduced pressure using a rotary evaporator to obtain the mycelium extract. Weigh 50 mg of the extract, dissolve it with chromatographic methanol, prepare a solution with a concentration of 5 mg / mL, filter it through a 0.22 μm filter membrane, and analyze it using high-performance liquid chromatography ( Figure 1 ).

[0046] The conditions for high-performance liquid chromatography analysis are as follows: The analytical chromatographic column is a YMC-Pack ODS-A series C18 chromatographic column (4.6 mm × 250 mm, 5 μm). Use 1‰ acetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution (0 - 40 min, B / A: 10% → 90%; 40 - 55 min, B / A: 90% → 100%; 55 - 75 min, B / A: 100% → 100%), the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL.

[0047] 2. Isolation of Demethylincisterol A3 in the Fermentation Product

[0048] Weigh 200 mg of the mycelium extract described in 1, dissolve it with 5 mL of ethanol, and then perform macroporous resin column chromatography separation. Use an ethanol / water system (60:40 - 100:0) for gradient elution, collect fractions in segments, and obtain 5 components. Use a high-performance liquid chromatograph to detect the fraction where Demethylincisterol A3 is located, combine all fractions containing Demethylincisterol A3, and perform reverse-phase column chromatography. Use a methanol / water system (70:30 - 100:0) for gradient elution. After high-performance liquid chromatography analysis, combine the fractions containing Demethylincisterol A3, and then purify it by semi-preparative high-performance liquid chromatography. Use an acetonitrile / water system (70 / 30) for isocratic elution to obtain 9.6 mg of the fermented Demethylincisterol A3 compound. Use high-performance liquid chromatography analysis for detection ( Figure 2 ).

[0049] The conditions for high-performance liquid chromatography analysis are as follows: The analytical chromatographic column is a YMC-Pack ODS-A series C18 chromatographic column (4.6 mm × 250 mm, 5 μm). Use a 1‰ acetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution (0 - 40 min, B / A: 10% → 90%; 40 - 55 min, B / A: 90% → 100%; 55 - 75 min, B / A: 100% → 100%), the flow rate is 1.0 mL / min, the column temperature is 30°C, and the injection volume is 10 μL.

[0050] 3. Identification of Fermented Demethylincisterol A3

[0051] Take the fermented Demethylincisterol A3 compound and perform structure identification by combining nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. The identification results are basically consistent with the standard Demethylincisterol A3 compound.

[0052] The conditions for nuclear magnetic resonance hydrogen spectrum analysis: Measure on a Bruker-ARX-400 nuclear magnetic resonance spectrometer, the solvent is deuterated chloroform, and the internal standard is tetramethylsilane.

[0053] 1 H-NMR (400 MHz, CDCl3) δ: 5.62 (1H, d, J =2, H-2), 5.25(1H, dd, J =15.2,7.6, H-16), 5.16 (1H, dd, J= 15.2, 8.8 Hz, H-15), 2.65 (1H, m, H-8), 2.29 (1H, m, H-5), 2.05 (1H, m, H-13), 1.97 (1H, m, H-6), 1.85 (3H, m, H-5, 10 and 17), 1.69 (2H, m, H-6 and 9), 1.48 (3H, m, H-10, 11 and 18), 1.00 (3H, d, J = 6.8 Hz, H-14), 0.92 (3H, d, J = 6.8 Hz, H-21), 0.83 (6H, d, H-19 and 20), 0.61 (3H, s, H-12).

[0054] 13 C-NMR (100 MHz, CDCl3) δ: 171.0 (C-1), 112.1 (C-2), 171.3 (C-3), 105.0 (C-4), 35.4 (C-5), 34.9 (C-6), 49.0 (C-7), 50.4 (C-8), 21.7 (C-9), 29.2 (C-10), 55.4 (C-11), 11.9 (C-12), 40.2 (C-13), 21.3 (C-14), 134.9 (C-15), 132.0 (C-16), 42.9 (C-17), 33.3 (C-18), 19.5 (C-19), 20.1 (C-20), 17.5 (C-21).

[0055] Example 3 Effect of Demethylincisterol A3 on the Expression of Antioxidant Signaling Pathway Proteins in Mouse Hepatocyte AML12

[0056] Digest, centrifuge and count AML12 cells, add the cell suspension into a 6-well plate, 2×10 per well 5 / mL. After the cells adhered to the wall, different concentrations of Demethylincisterol A3 were added and the cells were treated for 24 h. The culture medium was discarded, 1 mL of phosphate buffer solution (PBS) was added for washing, and after discarding the PBS, 200 μL of RIPA cell lysate was added. The cells were scraped and transferred to a 1.5 mL EP tube, lysed on ice for 30 min, mixed every 5 min, and then centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was transferred to a new 1.5 mL EP tube to obtain total protein, and Western blot was used for protein quantitative analysis.

[0057] The results are as Figure 5 and Figure 6 shown. As the concentration of Demethylincisterol A3 increased, the expression of HO-1 protein, p62, and the phosphorylation level of p62 in AML12 cells gradually increased, while the expression of Keap1 protein decreased. This suggests that in AML12 cells, Demethylincisterol A3 may activate the p62 protein, competitively bind to the Keap1 protein and mediate its degradation, release the oxidative stress-related transcription factor Nrf2 into the nucleus, initiate the expression of antioxidant proteins such as HO-1, resist cell peroxidation, and exert a hepatoprotective effect.

[0058] Example 4 Effect of Demethylincisterol A3 on the Transcription Level of Antioxidant Genes in Mouse Hepatocytes AML12

[0059] AML12 cells were digested, centrifuged, and counted. The cell suspension was added to a 6-well plate, 2×10 5 / mL. After the cells adhered to the wall, different concentrations of Demethylincisterol A3 were added and the cells were treated for 24 h. The culture medium was discarded, 1 mL of phosphate buffer solution (PBS) was added for washing, and after discarding the PBS, RNA special extraction solution was added. The extracted RNA was reverse transcribed into cDNA, and then real-time fluorescence quantitative PCR (RT-qPCR) analysis was performed to detect the changes in target genes.

[0060] The results showed that, as Figure 7 shown, compared with the blank control, as the concentration of Demethylincisterol A3 increased, the expressions of the antioxidant protein gene HO-1 and glutamate-cysteine ligase (GCLM) were extremely significantly increased, indicating that Demethylincisterol A3 can also induce the increase in the expression of antioxidant genes at the RNA level, thereby exerting an antioxidant effect.

[0061] Example 5 Evaluation of the Hepatoprotective Effect of Demethylincisterol A3 in a Mouse Model of Acute Liver Injury

[0062] To evaluate the protective effect of Demethylincisterol A3 in acute liver injury, an acute liver injury model was established by intraperitoneal injection of acetaminophen (APAP) in mice. NAC (acetaminophen + N-acetylcysteine), which is known to have a hepatoprotective effect, was selected as the positive control for treatment. Male C57BL / 6 mice aged 6 - 8 weeks were selected and randomly divided into a Control group, a DMA3 (Demethylincisterol A3) group, an APAP group, an APAP + NAC group, and an APAP + DMA3 group.

[0063] Control group: That is, the blank control group, intraperitoneally injected with the same volume of solvent; DMA3 group: 7 days before the experiment, DMA3 was intraperitoneally injected regularly every day, dissolved in olive oil, and APAP was not injected; APAP model group: After the mice were fasted for 12 h, APAP was intraperitoneally injected (the dosage was calculated according to the body weight of the mice, 400 mg / kg, the same below); APAP + NAC positive control group: After the mice were fasted for 12 h, APAP (400 mg / kg) was intraperitoneally injected, and NAC (150 mg / kg) was injected 2 h later; APAP + DMA3 experimental group: 7 days before the experiment, DMA3 (2 mg / kg) was intraperitoneally injected regularly every day. After the administration was completed, the mice were fasted for 12 h and then APAP (400 mg / kg) was injected.

[0064] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of hepatocyte injury, in the sera of mice in each group were detected. As Figure 8 shown, intraperitoneal injection of APAP could significantly increase the levels of ALT and AST in the serum at 24 h. After intervention with NAC and DMA3, the levels of the liver injury markers ALT and AST in the serum both decreased significantly, and the intervention effect of DMA3 was close to that of the positive hepatoprotective drug NAC, indicating that Demethylincisterol A3 has a very good protective effect on APAP-induced acute liver injury. In addition, the results of H&E section staining of the liver tissues of mice in each group were as Figure 9 shown. Consistent with the serological detection, the liver of mice with APAP-induced acute liver injury showed extensive necrosis. After treatment with NAC and Demethylincisterol A3, the necrosis of the liver tissue was significantly improved, further demonstrating the efficacy of Demethylincisterol A3 in drug-induced acute liver injury.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various changes and modifications in form and details can be made to the above embodiments without departing from the spirit and scope of the present invention, and all of them should be included in the protection scope defined by the claims of the present invention. Therefore, the protection scope of the present invention shall be subject to the claims.

Claims

1. A fungus, characterized in that, The fungus is a fungus of the family Cordycipitaceae Parengyodontium album SDGP-1; the fungus of the family Cordycipitaceae Parengyodontium album SDGP-1 is deposited in the China Center for Type Culture Collection, Wuhan, Hubei, with the deposit number CCTCC M 2025301 and the deposit date February 25, 2025.

2. Use of the fungus according to claim 1 in any one of the following: (1) Preparation of the natural product Demethylincisterol A3; (2) Preparation of an extract containing the natural product Demethylincisterol A3.

3. A method for preparing a natural product Demethylincisterol A3, characterized in that, Extract and isolate from the mycelium after fermentation using the fungus according to claim 1.

4. The preparation method of Demethylincisterol A3 according to claim 3, characterized in that, Comprising the following steps: (1) Inoculate the fungus SDGP-1 according to claim 1 into a seed medium for culture to obtain a seed solution of the strain SDGP-1; (2) Transfer the seed solution of the strain SDGP-1 to a fermentation medium for culture to obtain mycelium containing the natural product Demethylincisterol A3; (3) The mycelium containing the natural product Demethylincisterol A3 is filtered, and after separating the mycelium, it is dried, extracted, and concentrated under reduced pressure to obtain an extract of Demethylincisterol A3; (4) The extract of Demethylincisterol A3 is separated by resin, eluted with an alcohol solution, subjected to reverse-phase column chromatography, eluted with an alcohol solution, purified by high-performance liquid chromatography, and eluted with an acetonitrile solution to obtain the natural product Demethylincisterol A3.

5. The preparation method of Demethylincisterol A3 according to claim 4, characterized in that, The formulations of the said seed medium and fermentation medium are the same, and both are products obtained by boiling peeled potatoes and mixing them with glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and peptone, fully dissolving, making up the volume with water, and sterilizing.

6. Use of an extract of Demethylincisterol A3 or the natural product Demethylincisterol A3 prepared by the preparation method according to any one of claims 3-5 in the preparation of a medicament for treating acute liver injury, characterized in that, Comprising the method according to any one of claims 3-5.

7. The application according to claim 6, wherein The extract of Demethylincisterol A3 or the natural product Demethylincisterol A3 treats acute liver injury diseases by reducing the levels of alanine aminotransferase and aspartate aminotransferase in the serum.

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