Fungus, method for preparing Demodelincisterone A3 by using fungus and medicine for treating acute liver injury
By isolating the fungus Parengyodontium album SDGP-1 from the wild edible fungus Purple Gyros, and using fermentation methods, the yield of Demethylincisterol A3 was improved, the problem of low compound yield was solved, and large-scale preparation was achieved, which expanded the possibility for it in medicine and liver protection applications.
Patent Information
- Application Number
- CN202510601403.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-06-06
- Estimated Expiration
- 2045-05-12
AI Technical Summary
The yield of the compound Demethylincisterol A3 in the prior art is extremely low and cannot be prepared on a large scale, limiting its expansion in medicine and liver protection applications.
The fungus Parengyodontium album SDGP-1 was isolated from the wild edible fungus Purple Gyromite, and the yield of Demethylincisterol A3 was increased by fermentation, with the isolation yield reaching 4.8% or above.
The large-scale and continuous preparation of Demethylincisterol A3 has been achieved, providing important resources for its application areas such as liver protection and anti-cancer, and significantly improving its application potential in medicine.
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Abstract
Description
Technical Field
[0001] The present invention relates to a fungus Parengyodontium album , in particular, a fungus containing the compound Demethylincisterol A3 in fermented mycelium Parengyodontium album , as well as a culture method of the bacteria and a 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 demethylincisterol compound. Studies have shown that this compound has significant inhibitory activity against various cancer cell lines such as human breast cancer cell line MCF-7, colon cancer cell line HT-29, and bacteria such as Staphylococcus aureus, Escherichia coli, and Helicobacter pylori. It can also produce a good lethal effect on Artemia. In recent years, some researchers have isolated this compound from the secondary metabolites of various fungi, but the content is extremely low (<0.1%) and it is impossible to achieve large-scale preparation of this compound. Wild edible fungus Purple Gyrococcus [ Gomphus purpuraceus The compound is contained in the fruiting body of (Iwade) K. Yokoy., with a content of 0.001%, and is limited by seasonal production. The present invention isolates a fungus ( Parengyodontium album )SDGP-1, the mycelium contains DemethylincisterolA3, and the isolated yield is 4.8%, which is higher than the yield reported in the existing literature. It can be used for large-scale and continuous preparation and production of DemethylincisterolA3, providing an important resource for its application expansion.
[0003]
[0004] Compound Demethylincisterol A3 structural formula. Summary of the invention
[0005] The object of the present invention is to provide a fungus, which can produce active natural product Demethylincisterol A3 by fermentation or prepare an extract containing the natural product Demethylincisterol A3.
[0006] The present invention provides a fungus SDGP-1 ( Parengyodontium album ); the Cordyceps fungus SDGP-1 is deposited in China Center for Type Culture Collection, Wuhan, Hubei, with the deposit number CCTCC M 2025301 and the deposit date February 25, 2025.
[0007] In one aspect, the present invention provides a method for preparing the natural product Demethylincisterol A3, which is extracted and separated from mycelium after fermentation using the fungus, comprising the following steps: (1) inoculating the fungus SDGP-1 into a seed culture medium to obtain a seed solution of the strain SDGP-1; (2) The seed liquid of strain SDGP-1 was transferred to a fermentation medium to obtain mycelium containing the natural product Demethylincisterol A3; (3) The mycelium containing the natural product Demethylincisterol A3 is filtered, the mycelium is separated, and then dried, extracted, and concentrated under reduced pressure to obtain an extract of Demethylincisterol A3; (4) The extract of Demethylincisterol A3 was separated by resin, eluted with alcohol solution, chromatographed on a reversed phase column, eluted with alcohol solution, purified by high performance liquid chromatography, and eluted with acetonitrile solution to obtain the natural product Demethylincisterol A3.
[0008] The seed culture medium and the fermentation culture medium have the same formula, and are both products obtained by boiling peeled potatoes, mixing with glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and peptone to fully dissolve, adding water to make up the volume and sterilizing.
[0009] In some preferred cases, the fungus SDGP-1 is first inoculated into a sterilized seed culture medium, cultured in a constant temperature shaking incubator at 24°C (150r / min) for 14 days, and the seed liquid of the strain SDGP-1 is obtained. Then the seed liquid is transferred to a sterilized fermentation medium, and cultured in a constant temperature shaking incubator at 24°C (150r / min) for 14 days. The formula of the seed culture medium and the fermentation culture medium is the same, which is expressed as: peeled potatoes are cut into pieces and boiled for 30 minutes, the boiled juice is filtered, mixed with glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and peptone to fully dissolve, water is added to the fixed volume, the pH value is natural, and sterilized at 115°C for 30 minutes. The final fixed volume of the culture medium, calculated by 1L volume, contains the starting raw materials of 200g peeled potatoes, 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate heptahydrate, and 2g peptone. For the fermentation medium, the amount of raw materials can be expanded by multiples.
[0010] In some preferred cases, after the fungus SDGP-1 is fermented and cultured, the separation and preparation of Demethylincisterol A3 is carried out according to the following procedure: the fermentation broth is filtered to separate the mycelium, the mycelium is dried in a constant temperature drying oven, and then extracted with ethyl acetate for 3 times, and the extract is concentrated under reduced pressure by a rotary evaporator to obtain the extract of Demethylincisterol A3. The extract is separated by macroporous resin chromatography, gradient eluted with an ethanol / water system (60 / 40-100 / 0), and collected in sections to obtain 5 components. The components containing the target chemical are detected by a high performance liquid chromatography, and all the components containing the target compound are combined for reverse phase column chromatography, and gradient eluted with a methanol / water system (70 / 30-100 / 0). After high performance liquid chromatography analysis, the components containing the target compound are combined, and then purified by semi-preparative high performance liquid chromatography, and the target compound Demethylincisterol A3 is obtained by isogradient elution with an acetonitrile / water system (70 / 30).
[0011] The temperature of the constant temperature drying oven is 45-50°C.
[0012] The macroporous resin is D101. The macroporous resin column separation step is to perform gradient elution with an ethanol / water system (60 / 40-100 / 0), use high performance liquid chromatography to detect and combine the components containing Demethylincisterol A3, and obtain a macroporous resin column enriched fragment.
[0013] The reverse phase column filler is C18, and the chromatographic separation step is to perform gradient elution with a methanol / water system (70 / 30-100 / 0), and use high performance liquid chromatography to detect and combine the components containing Demethylincisterol A3 to obtain a reverse phase column enriched fragment.
[0014] The HPLC detection conditions are as follows: the chromatographic column is a YMC-Pack ODS-A series C18 chromatographic column (4.6 mm × 250 mm, 5 μm), 1‰ glacial acetic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, gradient elution is performed (0-40 min, B / A: 10%→90%; 40-55 min, B / A: 90%→100%; 55-75 min, B / A: 100%→100%), flow rate is 1.0 mL / min, column temperature is 30°C, and injection volume is 10 μL.
[0015] The semi-preparative HPLC purification conditions are acetonitrile / water system (70 / 30) isocratic elution and detection wavelength of 217 nm.
[0016] Another aspect of the present invention is to provide a liver protection health care product, characterized in that it comprises the extract of Demethylincisterol A3 or the natural product Demethylincisterol A3.
[0017] The liver protection health care product is used to increase the expression of antioxidant signaling pathway proteins in liver cells AML12, wherein the expression of antioxidant signaling pathway proteins in liver cells AML12 includes the increase of the expression of HO-1 protein, p62 and the phosphorylation level of p62 in AML12 cells.
[0018] The liver protection health care product is used to improve the transcriptional expression of liver cell AML12 antioxidant gene, and the transcriptional expression of liver cell AML12 antioxidant gene includes the increase of antioxidant protein gene HO-1 and glutamate-cysteine ligase expression.
[0019] A medicine for acute liver injury comprises the extract of Demethylincisterol A3 or the natural product Demethylincisterol A3.
[0020] The extract of Demethylincisterol A3 or the natural product Demethylincisterol A3 is used in pharmaceutical production to increase the levels of alanine aminotransferase and aspartate aminotransferase in serum in acute liver injury diseases.
[0021] The drug for acute liver injury is used for the treatment of acute liver injury diseases by intraperitoneal injection.
[0022] The present invention separates a fungus ( Parengyodontium album )SDGP-1, the mycelium contains Demethylincisterol A3, and the isolation yield is 4.8% and above, which can be used for large-scale and continuous preparation and production of Demethylincisterol A3, providing an important resource for its application expansion. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 :Liquid chromatogram of ethyl acetate extract of SDGP-1 mycelium.
[0024] Figure 2 :Demethylincisterol A3 liquid chromatography.
[0025] Figure 3 :Nuclear magnetic resonance identification of Demethylincisterol A3 compound from SDGP-1 fermentation.
[0026] H NMR analysis conditions: measured on a Bruker-ARX-400 NMR spectrometer, the solvent was deuterated chloroform, and the internal standard was tetramethylsilane.
[0027] Figure 4 :Nuclear magnetic resonance spectroscopy identification of Demethylincisterol A3 compound from SDGP-1 fermentation.
[0028] Figure 5 :Changes of antioxidant proteins in mouse hepatocytes (AML12) after treatment with different concentrations of Demethylincisterol A3 for 24 h.
[0029] Figure 6 :Grayscale analysis of antioxidant protein bands after different concentrations of Demethylincisterol A3 were applied to mouse hepatocytes (AML12) for 24 h.
[0030] Figure 7 :Changes of antioxidant genes in mouse hepatocytes (AML12) after 24 h exposure to different concentrations of Demethylincisterol A3.
[0031] Figure 8 : Changes of ALT and AST levels in serum of mice in different groups.
[0032] Fig. 9 : H&E staining results of liver sections of mice in different groups. DETAILED DESCRIPTION
[0033] In order to explain the technical scheme and technical purpose of the present invention, the present invention is further introduced below in conjunction with the accompanying drawings and specific implementation methods.
[0034] Example 1 Liquid fermentation of SDGP-1 1. Preparation of seed solution: Weigh 200g of peeled potatoes, cut into pieces and boil for 30min. After filtering the boiled juice, mix with 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate heptahydrate, and 2g peptone and fully dissolve. Add water to 1 L, and the pH value is natural. Dispense into 250mL conical flasks, 100mL of liquid culture medium per bottle, seal the conical flasks and place them in a high-pressure steam sterilizer at 115℃ for 30 minutes. After cooling, inoculate the fungus SDGP-1, shake and culture in a constant temperature shaking incubator at 24℃ and 150r / min for 14 days to obtain the seed liquid of SDGP-1.
[0035] 2. Preparation of fermentation broth: Weigh 4kg of peeled potatoes, cut into pieces and boil for 30 minutes. After filtering the boiled juice, mix with 400g glucose, 60g potassium dihydrogen phosphate, 30g magnesium sulfate heptahydrate, and 40g peptone to fully dissolve, add water to 20L, and the pH value is natural. Dispense into 3L conical bottles, 1.5L liquid culture medium per bottle, seal the conical bottles and place them in a high-pressure steam sterilizer at 115℃ for 30 minutes. After cooling, inoculate SDGP-1 seed liquid, shake and culture in a constant temperature shaking incubator at 24℃ and 150r / min for 14 days to obtain SDGP-1 fermentation liquid.
[0036] Example 2 Preparation and Identification of Demethylincisterol A3 in Fermentation Products 1. Pretreatment of fermentation products The SDGP-1 fermentation broth in Example 1 was filtered using a 120-mesh nylon mesh to separate the mycelium. The mycelium was dried in a constant temperature drying oven, crushed with a pulverizer, extracted with ethyl acetate three times, and the extract was concentrated under reduced pressure on a rotary evaporator to obtain a mycelium extract. 50 mg of the extract was weighed, dissolved with chromatographic methanol, prepared into a 5 mg / mL solution, filtered with a 0.22 μm filter membrane, and analyzed using high performance liquid chromatography ( Figure 1 ).
[0037] The HPLC analysis conditions were as follows: the analytical column was a YMC-Pack ODS-A series C18 column (4.6 mm × 250 mm, 5 μm), with 1‰ glacial acetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution (0-40 min, B / A: 10%→90%; 40-55 min, B / A: 90%→100%; 55-75 min, B / A: 100%→100%), flow rate 1.0 mL / min, column temperature 30°C, and injection volume 10 μL.
[0038] 2. Separation of Demethylincisterol A3 from fermentation products Weigh 200 mg of the mycelium extract described in 1, dissolve it in 5 mL of ethanol, and then perform macroporous resin column chromatography separation, perform gradient elution with an ethanol / water system (60:40-100:0), and collect in sections to obtain 5 components. Use a high-performance liquid chromatograph to detect the component where Demethylincisterol A3 is located, combine all the components containing Demethylincisterol A3 for reverse phase column chromatography, perform gradient elution with a methanol / water system (70:30-100:0), combine the components containing Demethylincisterol A3 after high-performance liquid chromatography analysis, and then purify it by semi-preparative high-performance liquid chromatography, and elute it with an acetonitrile / water system (70 / 30) to obtain 9.6 mg of fermented Demethylincisterol A3 compound, which is detected by high-performance liquid chromatography analysis ( Figure 2 ).
[0039] The HPLC analysis conditions were as follows: the analytical column was a YMC-Pack ODS-A series C18 column (4.6 mm × 250 mm, 5 μm), with 1‰ glacial acetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution (0-40 min, B / A: 10%→90%; 40-55 min, B / A: 90%→100%; 55-75 min, B / A: 100%→100%), flow rate 1.0 mL / min, column temperature 30°C, and injection volume 10 μL.
[0040] 3. Identification of fermented Demethylincisterol A3 The fermented Demethylincisterol A3 compound was used for structural identification by combining H NMR and C NMR, and the identification result was basically consistent with that of the standard Demethylincisterol A3 compound.
[0041] H NMR analysis conditions: measured on a Bruker-ARX-400 NMR spectrometer, the solvent was deuterated chloroform, and the internal standard was tetramethylsilane.
[0042] 1 H-NMR (400 MHz, CDCl 3 ) 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).
[0043] 13 C-NMR (100 MHz, CDCl 3 ) 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).
[0044] Example 3 Effect of Demethylincisterol A3 on the Expression of Antioxidant Signaling Pathway Proteins in Mouse Hepatocyte AML12 AML12 cells were digested, centrifuged and counted, and the cell suspension was added to a 6-well plate, with 2×10 5 / mL. After the cells adhered to the wall, different concentrations of Demethylincisterol A3 were added for 24 h, the culture medium was discarded, 1 mL of phosphate buffered saline (PBS) was added for washing, PBS was discarded, and 200 μL of RIPA cell lysis buffer 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 12000 rpm, 4 ℃, for 10 min. The supernatant was transferred to a new 1.5 mL EP tube to obtain total protein, and protein quantitative analysis was performed using Western blot.
[0045] The results are as follows Figure 5 and Figure 6 As shown in the results, with the increase of Demethylincisterol A3 concentration, the expression of HO-1 protein and p62 as well as the phosphorylation level of p62 in AML12 cells gradually increased, and the expression of Keap1 protein decreased. This suggests that in AML12 cells, Demethylincisterol A3 may activate p62 protein, competitively bind to Keap1 protein and mediate its degradation, release oxidative stress-related transcription factor Nrf2 into the cell nucleus, initiate the expression of antioxidant proteins such as HO-1, resist cell peroxidation, and exert liver protection effect.
[0046] Example 4 Effect of Demethylincisterol A3 on Transcription Levels of Antioxidant Genes in Mouse Hepatocyte AML12 Cells AML12 cells were digested, centrifuged and counted, and the cell suspension was added to a 6-well plate, with 2×10 5 / mL. After the cells adhered to the wall, different concentrations of Demethylincisterol A3 were added for treatment for 24 h, the culture medium was discarded, 1 mL of phosphate buffered saline (PBS) was added for washing, PBS was discarded, and RNA 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 changes in the target gene.
[0047] The results show that if Figure 7 As shown in the figure, compared with the blank control, with the increase of Demethylincisterol A3 concentration, the expression of antioxidant protein gene HO-1 and glutamate-cysteine ligase (GCLM) increased significantly, indicating that Demethylincisterol A3 can also induce the increase of antioxidant gene expression at the RNA level, thereby exerting an antioxidant effect.
[0048] Example 5 Evaluation of the liver protective effect of Demethylincisterol A3 in a mouse model of acute liver injury In order to evaluate the protective effect of Demethylincisterol A3 in acute liver injury, the acute liver injury model was established by intraperitoneal injection of acetaminophen (APAP) in mice, and NAC (acetaminophen + N-acetylcysteine), which is known to have liver protective effects, was selected as a positive control for treatment. Male C57BL / 6 mice aged 6-8 weeks were selected and randomly divided into the Control group, DMA3 (Demethylincisterol A3) group, APAP group, APAP+NAC group, and APAP+DMA3 group.
[0049] Control group: blank control group, intraperitoneally injected with the same volume of solvent; DMA3 group: DMA3 dissolved in olive oil was intraperitoneally injected regularly every day for 7 days before the experiment, and APAP was not injected; APAP model group: after the mice were fasted for 12 hours, APAP was intraperitoneally injected (the dosage was calculated based on the weight of the mice, 400 mg / kg, the same below); APAP+NAC positive control group: after the mice were fasted for 12 hours, APAP (400 mg / kg) was intraperitoneally injected, and NAC (150 mg / kg) was injected 2 hours later; APAP+DMA3 experimental group: DMA3 (2 mg / kg) was intraperitoneally injected regularly every day for 7 days before the experiment. After the administration, the mice were fasted for 12 hours before being injected with APAP (400 mg / kg).
[0050] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of hepatocyte damage, in the serum of each group of mice were detected. Figure 8 As shown in the figure, intraperitoneal injection of APAP can significantly increase the levels of ALT and AST in serum within 24 hours. After intervention with NAC and DMA3, the levels of ALT and AST, the liver injury markers in serum, decreased significantly, and the intervention effect of DMA3 was close to that of the positive liver protection drug NAC, indicating that Demethylincisterol A3 has a very good protective effect on APAP-induced acute liver injury. In addition, the H&E section staining results of the liver tissues of each group of mice are shown in the figure. Fig. 9 As shown, consistent with serological tests, mice with APAP-induced acute liver injury had large areas of necrosis in the liver. After treatment with NAC and Demethylincisterol A3, the liver tissue necrosis was significantly improved, further proving the efficacy of Demethylincisterol A3 for drug-induced acute liver injury.
[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not used 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 should be included in the protection scope defined by the claims of the present invention. Therefore, the protection scope of the present invention should be based on the claims.
Claims
1. A fungus, characterized in that The fungus is a Cordyceps fungus ( Parengyodontium album ) SDGP-1; the Cordyceps fungus SDGP-1 is preserved in the China Center for Type Culture Collection, Wuhan, Hubei, with the preservation number CCTCCM 2025301 and the preservation date February 25, 2025.
2. The fungus of claim 1 is used in any of the following: (1) Preparation of natural product Demethylincisterol A3; (2) Preparation of extracts containing the natural product Demethylincisterol A3.
3. A method for preparing a natural product Demethylincisterol A3, characterized in that: The fungus according to claim 1 is used for fermentation and then extracted and separated from the mycelium.
4. The method for preparing Demethylincisterol A3 according to claim 3, characterized in that: The steps include: (1) inoculating the fungus SDGP-1 described in claim 1 into a seed culture medium to obtain a seed solution of the strain SDGP-1; (2) The seed liquid of strain SDGP-1 was transferred to a fermentation medium to obtain mycelium containing the natural product Demethylincisterol A3; (3) The mycelium containing the natural product Demethylincisterol A3 is filtered, the mycelium is separated, and then dried, extracted, and concentrated under reduced pressure to obtain an extract of Demethylincisterol A3; (4) The extract of Demethylincisterol A3 was separated by resin, eluted with alcohol solution, chromatographed on a reversed phase column, eluted with alcohol solution, purified by high performance liquid chromatography, and eluted with acetonitrile solution to obtain the natural product Demethylincisterol A3.
5. The method for preparing Demethylincisterol A3 according to claim 4, characterized in that: The seed culture medium and the fermentation culture medium have the same formula, and are both products obtained by boiling peeled potatoes, mixing with glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate and peptone to fully dissolve, adding water to make up the volume and sterilizing.
6. A liver protection health product, characterized in that: The invention relates to an extract of Demethylincisterol A3 or a natural product Demethylincisterol A3 as claimed in claim 4 or 5.
7. The liver protection health product according to claim 6, characterized in that: The liver protection health care product is used to increase the expression of antioxidant signaling pathway proteins in liver cells AML12, wherein the expression of antioxidant signaling pathway proteins in liver cells AML12 includes the increase of the expression of HO-1 protein, p62 and the phosphorylation level of p62 in AML12 cells.
8. The liver protection health product according to claim 6, characterized in that: The liver protection health care product is used to improve the transcriptional expression of liver cell AML12 antioxidant genes, and the transcriptional expression of liver cell AML12 antioxidant genes includes the increase of the expression of antioxidant protein gene HO-1 and glutamate-cysteine ligase (GCLM).
9. A drug for acute liver injury, characterized in that: The invention relates to an extract of Demethylincisterol A3 or a natural product Demethylincisterol A3 as claimed in claim 4 or 5.
10. The drug for acute liver injury according to claim 9, characterized in that: The extract of Demethylincisterol A3 or the natural product Demethylincisterol A3 is used in pharmaceutical production to increase the levels of alanine aminotransferase and aspartate aminotransferase in serum in acute liver injury diseases.
Citation Information
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