Pyranopyridone alkaloid compound as well as preparation method and application thereof

Abstract

The invention discloses a pyranopyridone alkaloid compound as well as a preparation method and application thereof, and belongs to the technical field of medicines. The pyranopyridone alkaloid compound with the activity of resisting the acetaminophen-induced acute liver injury is extracted from a fermentation product of a marine fungus Aspergillus aculeatusWHUF0198, and the pyranopyridone alkaloid compound improves the cell survival rate of mouse hepatocytes treated by acetaminophen and reduces the glutamic-pyruvic transaminase level in a cell supernatant. The pyranopyridone alkaloid compound has the advantages that the pyranopyridone alkaloid compound has the activity of resisting the acetaminophen-induced acute liver injury, and the pyranopyridone alkaloid compound has the activity of resisting the acetaminophen-induced acute liver injury; the pyran pyridone alkaloid compound disclosed by the invention can be used for preparing a medicine for treating acute liver injury. Aiming at the current situation that the types of medicines for treating acute liver injury caused by acetaminophen are deficient at present, the traditional Chinese medicine composition is rich in potential economic benefit.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a pyranopyridone alkaloid compound separated from a fermentation product of Aspergillus pseudoacanthus fungus, a preparation method thereof, and an application thereof in preparing a medicine for treating acute liver injury. Background Art

[0002] Acute liver injury (ALI) is characterized by symptoms such as hepatocellular damage, necrosis, apoptosis, and liver dysfunction that occur in a short period of time. ALI can be caused by a variety of factors, including drugs, viral infections, liver surgery, and chemical toxins. Drug-induced liver injury (DILI) is the main cause of ALI, accounting for more than 50% of cases. DILI refers to liver damage caused by various drugs and their metabolites or excipients. Drugs that often cause DILI are acetaminophen (APAP), amiodarone, steroids, antimetabolites, valproic acid, niacin, statins, etc. Among them, DILI caused by the combined use of APAP and isoniazid and rifampicin is the most common. The main pathogenesis is the compensatory inactivation of the antioxidant stress system and the accumulation of ROS, which in turn leads to damage to the mitochondrial function of hepatocytes.

[0003] APAP-induced ALI develops suddenly and is often accompanied by liver failure, hepatic encephalopathy, and other serious complications, leading to severe oxidative stress and persistent inflammatory response. If not treated in time, it may lead to death. Therefore, it is of great significance to develop new drugs with multiple targets and multiple action pathways to treat APAP-induced ALI. N-acetyl-L-cysteine ​​(NAC) is the most commonly used drug for the treatment of APAP poisoning in clinical settings, but its use is complicated by its narrow dosing window and potential serious side effects. Therefore, it is of great clinical significance to find more effective drugs with fewer side effects to treat APAP-induced liver injury.

[0004] Pyranopyridone alkaloids are a class of alkaloid compounds with a 2-pyridone-pyran skeleton. These compounds from natural sources and artificial synthesis have a wide range of biological activities, such as anti-tumor, antibacterial, antifungal and anti-inflammatory activities. Currently, only 14 pyranopyridone compounds with unsaturated side chains on the pyran ring have been reported in the literature. They have Ca 2+Inhibitory effect, NO release inhibition effect. Due to their unique biological activity, this class of compounds has received great attention from synthetic and medicinal chemists. However, the current research scope of pyranopyridone alkaloids with unsaturated side chains is quite limited, and there are no reports on the ALI-improving activity of such compounds. Therefore, it is of great research value to explore pyranopyridone derivatives with ALI-improving activity from natural sources. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the present invention aims to provide a pyranopyridone alkaloid compound having anti-APAP-induced acute liver injury activity extracted from the fermentation product of the marine fungus Aspergillus aculeatinus WHUF0198, and a preparation method and pharmaceutical application thereof. The pyranopyridone alkaloids 1-7, 9, 10, 12-15 provided by the present invention can improve the cell survival rate of AML-12 cells (mouse hepatocytes) treated with APAP, and reduce the level of alanine aminotransferase (ALT) in the cell supernatant, and have the potential to be developed into a drug for treating acute liver injury induced by APAP. The present invention provides a lead compound for the development of drugs for treating DILI.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The invention obtains 17 pyranopyridone alkaloid compounds by culturing marine fungus Aspergillus aculeatinus WHUF0198 alone and co-culturing it with mangrove-derived fungus Penicillium sp. DM27 fungus, and extracting them from fermentation products.

[0008] The pyranopyridone alkaloid compound provided by the present invention is compound 1-17 shown in the following structural formula.

[0009] The structural formula of compounds 1, 2, 3, and 4:

[0010] The group combinations of compounds 1, 2, 3, and 4 are shown in the following table:

[0011] serial number <![CDATA[R1]]> <![CDATA[R2]]> Compound 1 <![CDATA[CH3]]> <![CDATA[CH3]]> Compound 2 <![CDATA[CH2OH]]> <![CDATA[CH3]]> Compound 3 COOH <![CDATA[CH3]]> Compound 4 <![CDATA[CH3]]> COOH

[0012] The structural formulas of compounds 5, 6, 7, 8, 9, 10 and 11 are:

[0013] The group combinations of compounds 5, 6, 7, 8, 9, 10, and 11 are shown in the following table:

[0014]

[0015]

[0016] The structural formulas of compounds 12, 13, 14 and 15 are:

[0017] The group combinations of compounds 12, 13, 14, and 15 are shown in the following table:

[0018] serial number <![CDATA[R1]]> <![CDATA[R2]]> Compound 12 <![CDATA[CH2OH]]> <![CDATA[CH3 <!-- 2 -->]]> Compound 13 COOH <![CDATA[CH3]]> Compound 14 <![CDATA[CH3]]> COOH Compound 15 <![CDATA[CH2OCHO]]> <![CDATA[CH3]]>

[0019] The structural formula of compound 16:

[0020] The structural formula of compound 17:

[0021] The preparation method of the pyranopyridone alkaloid compound comprises the following steps: inoculating Aspergillusaculeatinus WHUF0198 alone or simultaneously with Penicillium sp.DM27 into a liquid culture medium for separate fermentation or co-fermentation, and separating the pyranopyridone alkaloid compound from the fermentation product.

[0022] Furthermore, the preparation method of the pyranopyridone alkaloid compound comprises the following steps: inoculating Aspergillus aculeatinus WHUF0198 alone or simultaneously inoculating it with Penicillium sp.DM27 into a liquid culture medium for single fermentation or co-culture fermentation, separating the fermentation broth from the mycelium, crushing the mycelium and extracting it with acetone ultrasonically, recovering the acetone and concentrating it under reduced pressure, and then combining it with the fermentation broth. The combined solution is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract; or the combined solution is added with a macroporous resin for adsorption, and the macroporous resin adsorbing the metabolites is subjected to column chromatography and gradient elution with methanol / water to obtain components containing different methanol contents, using HPLC-DAD to detect the characteristic absorption peak of the pyranopyridone alkaloid, combining LC-MS to analyze the presence of nitrogen atoms, and determining the component where the pyridine pyrone alkaloid is located, and these components are combined and concentrated under reduced pressure to obtain a crude extract. Then, the crude extract is separated by silica gel column chromatography to obtain smaller components, and then separated by Sephadex LH-20 gel column chromatography. The characteristic peaks of pyranopyridone alkaloid compounds are purified by semi-preparative liquid phase to obtain pyranopyridone alkaloid compounds.

[0023] The pyranopyridone alkaloid compound has an effect of resisting acute liver injury. Based on this, the present invention also provides the use of the pyranopyridone alkaloid compound in the preparation of a drug for treating acute liver injury. The pyranopyridone alkaloid compound comprises one or more of the above compounds 1-17. Further, the acute liver injury is drug-induced liver injury. Further, the acute liver injury is acetaminophen-induced acute liver injury.

[0024] The present invention also provides a drug for treating acute liver injury, which comprises the above-mentioned pyranopyridone alkaloid compound and may further comprise a pharmaceutically acceptable excipient; the pyranopyridone alkaloid compound comprises one or more of the above-mentioned compounds 1-17.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] First, the culture conditions for the single culture of the strain Aspergillus aculeatinus WHUF0198 and the co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27 used in the present invention are easy to control and have good reproducibility. A sufficient amount of pyranopyridone alkaloid compounds can be obtained through fermentation, thereby solving the problem that there are few natural source acquisition channels for such compounds and the artificial synthesis steps are cumbersome.

[0027] Secondly, the preparation method of the alkaloid compound of the present invention comprises: extracting mycelium by ultrasonic acetone, extracting fermentation liquid by adsorption with macroporous resin HP20, eluting with gradient solvent with macroporous resin column, silica gel column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography separation and purification, etc. The materials and equipment required for separation are simple and common, and the purification process is efficient and feasible.

[0028] Thirdly, the pyranopyridone alkaloids separated and prepared by the present invention have a significant repairing effect on mouse liver cell damage caused by acetaminophen, and can be used to prepare drugs for treating acute liver injury caused by acetaminophen. Considering the current situation of a shortage of drugs for treating acute liver injury caused by acetaminophen, the potential economic benefits are huge. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the cell survival rate of normal mouse hepatocytes AML-12, APAP-induced liver injury AML-12 cells, and APAP-induced liver injury cells treated with different concentrations of compounds 1-7 and 9-16.

[0030] Figure 2The above is the level of alanine aminotransferase (ALT) in the supernatant of normal mouse hepatocytes AML-12, APAP-induced liver injury AML-12 cells, and APAP-induced liver injury cells treated with compounds 1-7 and 9-16. (c=5μM, n=3-6). The data are expressed as mean SD. All compounds significantly reduced the ALT level *P<0.05, **P<0.01, ***P<0.001.

[0031] Figure 3 It is a Venn diagram of acute liver injury (ALI) disease-related targets and compound 12-related targets.

[0032] Figure 4 This is the GO analysis of compound 12 targets.

[0033] Figure 5 This is the KEGG analysis of compound 12 targets.

[0034] Figure 6 It is a network diagram of ALI-related binding sites of compound 12. The color depth represents the degree of association.

[0035] Figure 7 is the binding energy of compound 12 with the core target protein.

[0036] Figure 8 Molecular docking of compound 12 and ALI-related target proteins: (A) AKT, (B) HSP90AA1, (C) MTOR, (D) BCL2, (E) PIK3CA, (F) STAT3, (G) SRC, (H) TNF-α, (I) GRB2, (J) EGFR. DETAILED DESCRIPTION

[0037] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0038] Example 1: Preparation of pyranopyridone alkaloid compounds

[0039] A. Strain fermentation

[0040] Strain Aspergillus aculeatinus WHUF0198 was fermented separately: Aspergillus aculeatinus WHUF0198 (the strain has been published in the following articles: Wu J, Zhang H, He LM, et al, A New Fusicoccane-Type Norditerpene and a New Indone from the Marine-Derived Fungus Aspergillus aculeatinus WHUF0198 [J]. Chemistry & Biodiversity. 2021, 18 (10): e2100562; Wu J, Wang F, He LM, et al, Aculeaquamide A, Cytotoxic Paraherquamide from the Marine Fungus Aspergillus Aculeatinus WHUF0198 [J]. Natural Product Research. 2022, 36 (17), 4382–4387; Wu J, Shui H, Zhang M, et al. al.Aculeaxanthones A–E, New Xanthones from the Marine-Derived Fungus Aspergillus Aculeatinus WHUF0198[J].Frontiers in Microbiology.2023,14.) were inoculated in a culture dish containing PDA solid culture medium, and cultured at 28°C in a biochemical incubator for 7 days. The solid culture medium was then divided into 1cm×1cm blocks and inoculated into a 1L conical flask containing 300mL PDB liquid culture medium for fermentation. The fermentation temperature was 28°C, the fermentation time was 28 days, and the total fermentation volume was 100 bottles. PDA solid culture medium formula: 26g potato glucose water (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), 20g agar, 1000mL water, pH 6.5-7; PDB liquid culture medium formula: 26g potato glucose water (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), 15g sea salt, 1000mL water, pH 6.5-7.

[0041] Co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27: Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27 (the strain has been published in the following articles: He LM, Zhao ZF, Zhang MK, et al, Peniciisoquinoline A: A newtetrahydroisoquinoline from mangrove-derived Penicillium sp. DM27 fungus [J]. Journal of Holistic Integrative Pharmacy. 2022, 3(3): 222-227; Dong Zhitong, Isolation and screening of two special habitat fungi and study of the metabolites of Aspergillus pyrococcus 094102 [D]. Wuhan University, 2017.) were inoculated into culture dishes containing PDA solid culture medium, and cultured at 28°C in a biochemical incubator for 7 days. Then, the solid culture medium was divided into 1 cm × 1 cm blocks and cultured according to the Aspergillus aculeatinus WHUF0198:Penicillium sp.DM27=1:3 ratio, inoculated into 1L conical flask containing 300mL PDB liquid medium for fermentation, fermentation temperature was 28℃, fermentation time was 28 days, total fermentation volume was 100 bottles. Medium formula was the same as single culture.

[0042] B. Fermentation product extraction

[0043] After the fermentation time is reached, the fermentation liquid and mycelium in the conical flask are separated by filter cloth, the mycelium is chopped into small pieces and completely immersed in acetone, placed in an ultrasonic cleaning machine, ultrasonicated for 30 minutes, the mycelium is stirred after the ultrasonication, and ultrasonicated again, and repeated 3 times. The acetone extract and mycelium are filtered to separate, the mycelium is immersed in acetone again, and the above ultrasonic extraction steps are repeated. The acetone extract and mycelium are filtered again, and the acetone extract obtained by the two ultrasonic extractions are combined, the reduced pressure evaporation is concentrated, the acetone is recovered, and the concentrate is combined with the fermentation liquid. The above steps are applicable to the extraction of fermentation products of Aspergillus aculeatinus WHUF0198 alone and Aspergillus aculeatinus WHUF0198 and Penicillium sp.DM27 co-culture.

[0044] The fermentation pooled liquid of Aspergillus aculeatinus WHUF0198 fermented alone was extracted three times with equal amounts of ethyl acetate (EA), and then the solvent of the EA layer was evaporated under reduced pressure to obtain 18.0 g of a dry crude extract.

[0045] For the fermentation combined liquid of Aspergillus aculeatinus WHUF0198 and Penicillium sp.DM27 co-cultured, 2L HP20 macroporous resin was added to the combined liquid and stirred for 8 hours for secondary metabolite adsorption and extraction. After stirring, the macroporous resin adsorbed with the secondary metabolites of the strain was filtered. The macroporous resin was loaded into a glass chromatography column and then eluted with five different proportions of methanol aqueous solution of 30%, 50%, 70%, 90%, and 100%, and the volume of each proportion of eluent was 5L. After elution, the 70%, 90%, and 100% eluents were combined, and the solvent was evaporated under reduced pressure to obtain 10.1g of dry crude extract.

[0046] C. Separation and preparation of pyridopyrone alkaloid compounds.

[0047] For the crude extract of Aspergillus aculeatinus WHUF0198 fermented alone. 200-300 mesh normal silica gel was used as filler, silica gel column chromatography (CC), and dichloromethane / methanol (DCM / MeOH) was used for elution and separation (volume ratio gradient was 80:1, 40:1, 20:1, 10:1, 5:1 and 1:1), and the eluted sample was received in a 250 mL receiving bottle. Silica gel thin layer chromatography (TLC) analysis was performed on each bottle of sample. A straight line was drawn with a pencil at a distance of 1 cm from the lower edge of the silica gel plate, and a capillary glass tube (0.3 mm) was used to absorb each bottle of solution, and points were placed on different positions on the straight line from left to right in turn, evenly distributed, and the sample number was marked with a pencil after drying for subsequent merging. Prepare a suitable ratio of developing agent, the developing agent system is DCM: MeOH (0.1% formic acid) = 100: 1-15: 1, so that the components in each sample are clearly separated after being developed on the silica gel plate and preferably located near the middle of the plate. Place the spotted silica gel plate into a developing cylinder filled with developing agent, with the spotted side facing down. Take out the thin layer plate after the developing agent is developed to 1 cm from the upper edge. Place the thin layer plate in a UV detection box and observe the development under 254 and 365nm UV lamps. Combine the samples with almost identical sample points after development, and spin dry them after merging to obtain six main components (Fr.1-6) in turn. The TLC analysis described below is all performed in accordance with the above content. The effect of each component on cell survival rate was evaluated using mouse hepatocytes induced by APAP at a concentration of 100μg / mL. The results showed that the cell death rates of Fr.5 and Fr.6 were significantly reduced compared with the non-administered group. These two components were further separated. Fr.5 (2.2 g) was separated by silica gel column chromatography, and the eluent gradient was DCM / EA, 20:1, 15:1, 10:1, 5:1, 0:1; EA / MeOH, 10:1, 5:1, 2:1, and 26 subfractions (Fr.5.a-Fr.5.z) were obtained by TLC analysis. At a concentration of 100 μg / mL, the cell survival rate of mouse hepatocytes induced by APAP was tested, and Fr.5.k (256 mg) showed a significantly reduced cell death rate compared with the untreated group. The component was then prepared using semi-preparative liquid chromatography (HPLC) with a Sepax GP-C-18 column (5 μm, 10×250 mm, used in the preparation process) and a mobile phase of 60-100% (V:V) methanol / water at a flow rate of 3 mL / min for 30 min to separate compounds 1 (3 mg, retention time 15.2 min) and 5 (3 mg, retention time 22.3 min).The Fr.6 component (3.7 g) was separated by silica gel column chromatography, using an eluent gradient of EtOAc / MeOH (20:1, 15:1, 10:1, 5:1, 0:1, V / V), and each eluted component was analyzed by TLC and combined to obtain 20 sub-fractions (Fr.6.a-Fr.6.t). The effect of each component on the survival rate of mouse hepatocytes induced by APAP was tested, and the cell death rate shown by Fr.6.c and Fr.6.f was significantly reduced compared with the non-administered group. Fr.6.c (408 mg) was separated by gel column chromatography, the filler was Sephadex LH-20 (this filler was used for gel column chromatography), and eluted with DCM / MeOH (1:1, V / V), and each eluted component was analyzed by TLC and combined to obtain 10 sub-fractions (Fr.6.c.1-10). Fr.6.c.5 (26 mg) was prepared by semi-preparative HPLC, and the mobile phase conditions were: 70-80% (V:V) methanol / water eluted at a flow rate of 3 mL / min for 30 min, and compound 6 (2 mg, retention time of 24.3 min) was separated. Under the same chromatographic conditions, semi-preparative HPLC was used to separate compounds 8 (8 mg, retention time of 18.6 minutes), 9 (2 mg, retention time of 20.3 min) and 10 (6 mg, retention time of 1.6.7 min) from Fr.6.c.6 (35 mg). Under the same chromatographic conditions, Fr.6.c.10 (19 mg) was prepared to obtain compound 17 (1 mg, retention time of 15.8 min). Fr.6.f (270 mg) was prepared, and the mobile phase conditions were: 70-100% (V:V) methanol / water eluted at a flow rate of 3 mL / min for 30 min, and 16 (4 mg, retention time of 17.6 min) was obtained.

[0048] For the crude extract (10.1 g) of the co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp.DM27, 200-300 mesh normal silica gel was used as the filler, column chromatography (CC), and dichloromethane / methanol (DCM / MeOH) (volume ratio gradient was 100:1, 40:1, 20:1, 10:1 and 5:1) was used to elute and separate the crude extract, and the eluted sample was received in a 250 mL receiving bottle. Each bottle of sample was subjected to silica gel thin TLC analysis and combined to obtain 5 subfractions (Fr.B.1-Fr.B.5). Fr.B.2 (468 mg) was separated by gel column chromatography and eluted with DCM / MeOH (1:1, V / V) to obtain three subfractions (Fr.B.2.1-Fr.B.2.3). Fr.B.2.2 (50 mg) was semi-prepared, and the mobile phase conditions were: 70-80% (V: V, 0.1% formic acid) methanol / water eluted at a flow rate of 3 mL / min for 40 min to obtain compound 12 (11 mg, retention time of 12.9 min). Fr.B.2.3 (70 mg) was semi-prepared, and the mobile phase conditions were: 40-70% (V: V, 0.1% formic acid) acetonitrile / water eluted at a flow rate of 3 mL / min for 40 min, and compound 13 (2 mg, retention time of 16.8 min), compound 14 (2 mg, retention time of 15.5 min), and compound 15 (2 mg, retention time of 1.23 min) were obtained at different retention times. Using gel column chromatography and TLC analysis, Fr.B.4 (1.03 g) was divided into seven subcomponents (Fr.B.4.1-Fr.B.4.4). Fr.B.4.3 (331 mg) was separated into five subcomponents (Fr.B.4.3.1-Fr.B.4.3.5) by silica gel column chromatography with a gradient of DCM / MeOH, 500:1, 100:1, 80:1, 50:1, 10:1, 5:1, and then combined with TLC analysis. Compound 11 (3 mg, retention time 33.6 min) was obtained from Fr.B.4.3.1 (21 mg) by semi-preparative HPLC with mobile phase conditions of 70-80% (V:V, 0.1% formic acid) methanol / water at a flow rate of 3 mL / min for 40 min. Semi-preparative high performance liquid chromatography was used with the mobile phase conditions of 65% (V:V) methanol / water at a flow rate of 3 mL / min for 40 min to obtain compounds 6 (2 mg, retention time 34.3 min), 9 (2 mg, retention time 30.7 min) and 10 (4 mg, retention time 26.5 min) from Fr.B.4.3.5 (21 mg).Fr.B.4.4 (96 mg) was separated by silica gel column chromatography, the eluent gradient was DCM / MeOH, 40:1, 20:1, 10:1, and the eluted components were analyzed by TLC and combined to obtain three subcomponents (Fr.B.4.4.1-Fr.B.4.4.3). Semi-preparative HPLC was used, and the mobile phase conditions were: 60-100% (V:V, 0.1% formic acid) methanol / water at a flow rate of 3 mL / min for 20 min, and compound 3 (2 mg, retention time of 13.1 min) and compound 4 (2 mg, retention time of 13.8 min) were obtained from Fr.B.4.4.2 (26 mg). Compound 2 (2 mg, retention time 24.6 min) was purified from Fr.B.4.4.3 (38 mg) by semi-preparative HPLC with a mobile phase of 30-50% (V:V, 0.1% formic acid) acetonitrile / water at a flow rate of 3 mL / min for 40 min. Fr.B.5 (324 mg) was separated by gel column and combined by TLC analysis to obtain six subcomponents (Fr.B.5.1-Fr.B.5.6). Compound 7 (3.5 mg, retention time 22.1 min) was isolated from Fr.B.5.4 (50.3 mg) by semi-preparative HPLC with a mobile phase of 40-80% (V:V, 0.1% formic acid) acetonitrile / water at a flow rate of 3 mL / min for 30 min.

[0049] C. Structural Elucidation

[0050] Each compound was analyzed by NMR, mass spectrometry and other methods, and the results are shown below.

[0051] Compound 1: yellow powder; [α] 2 D 0 -36 (c 0.02, MeOH); 1 H and 13 C NMR data are shown in Table 1; HR-ESI-MS (m / z): 328.1932 [MH] - (calcd for C 20 H 26 NO3,328.2277); ECD(c 21mM,MeOH),λ max (Δε)296(-0.40),275(+3.01),243(-2.79),228(+1.33),213(-37.14)nm.

[0052] Compound 2: white powder; [α] 2 D 0 -54 (c 0.04, MeOH); 1H and 13 C NMR data are shown in Table 1; HR-ESI-MS (m / z): 344.1857 [MH] - (calcd for C 20 H 26 NO4,344.1862); ECD(c 2.9mM,MeOH),λ max (Δε)295(-3.14),275(+2.98),245(+0.16),226(+1.84),212(-21.62)nm.

[0053] Compound 3: white powder; [α] 2 D 0 -57 (c 0.04, MeOH); 1 H and 13 C NMR data are shown in Table 1; HR-ESI-MS (m / z): 358.1661 [MH] - (calcd for C 20 H 24 NO5,358.1655); ECD(c 2.8mM,MeOH),λ max (Δε)298(-0.41),268(+4.70),243(-2.35),228(+2.17),213(-50.68)nm.

[0054] Compound 4: white powder; [α] 2 D 0 -12 (c 0.13, MeOH); 1 H and 13 C NMR data are shown in Table 2; HR-ESI-MS (m / z): 358.1652 [MH] - (calcd for C 20 H 24 NO5,358.1655); ECD(c 2.2mM,MeOH),λ max (Δε)294(-2.35),271(+2.31),245(-2.22),230(+1.59),215(-35.95)nm.

[0055] Compound 5: white powder; [α] 2 D 0 -19.5 (c 0.07, MeOH); 1 H and 13C NMR data see 2; HR-ESI-MS (m / z): 366.2038 [M+Na] + (calcd for C 21 H 29 NO3Na,366.2045); ECD(c 17mM,MeOH),λ max (Δε)275(+0.72),245(-4.09),228(+3.50),215(-30.28)nm.

[0056] Compound 6: white powder; [α] 2 D 0 -9 (c 0.01, MeOH); 1 H and 13 C NMR data are shown in Table 2; HR-ESI-MS (m / z): 382.1981 [M+Na] + (calcd for C 21 H 29 NO4Na,382.1994); ECD(c 16mM,MeOH),λ max (Δε)293(-1.13),264(+0.20),246(-1.06),229(+1.49),214(-16.80)nm.

[0057] Compound 7: yellow powder; [α] 2 D 0 -31.5 (c 0.10, MeOH); 1 H and 13 CNMR data are shown in Table 3; HR-ESI-MS (m / z): 398.1944 [M+Na] + (calcd for C 21 H 29 NO5Na,398.1943); ECD(c 3.6mM,MeOH),λ max (Δε)300(-1.12),276(+5.44),242(-1.53),229(+0.78),215(-30.75)nm.

[0058] Compound 8: white powder; [α] 2 D 0 -12 (c 0.03, MeOH); 1 H and 13C NMR data are shown in Table 3; HR-ESI-MS (m / z): 396.1778 [M+Na] + (calcd for C 21 H 27 NO5Na,396.1787); ECD(c 10mM,MeOH),λ max (Δε)267(+5.14),242(-1.53),229(+0.78),215(-30.71)nm.

[0059] Compound 9: white powder; [α] 2 D 0 -27 (c 0.13, MeOH); 1 H and 13 C NMR data are shown in Tables 3 and 4; HR-ESI-MS (m / z): 396.1778 [M+Na] + (calcd for C 21 H 27 NO5Na,396.1787); ECD(c 16mM,MeOH),λ max (Δε)293(-4.57),270(-2.05),246(-2.93),229(+3.08),213(-26.68)nm.

[0060] Compound 10: white powder; [α] 2 D 0 -12 (c 0.13, MeOH); 1 H and 13 C NMR data are shown in Table 4; HR-ESI-MS (m / z): 396.1772 [M+Na] + (calcd for C 21 H 27 NO5Na,396.1787); ECD(c 20mM,MeOH),λ max (Δε)292(-1.11),273(+0.86),244(-0.91),229(+1.48),215(-15.7)nm.

[0061] Compound 11: white powder; [α] 2 D 0 -4 (c 0.04, MeOH); 1 H and 13C NMR data are shown in Table 4; HR-ESI-MS (m / z): 388.2103 [M+H] + (calcd for C 22 H 30 NO5,388.2124); ECD(c 2.6mM,MeOH),λ max (Δε)295(-1.09),274(+2.86),244(-1.56),229(+0.17),215(-22.91)nm.

[0062] Compound 12: yellow oil; [α] 2 D 0 -18 (c 0.07, MeOH); 1 H and 13 C NMR data are shown in Table 5; HR-ESI-MS (m / z): 376.2118 [M+H] + (calcd for C 21 H 30 NO5,376.2124); ECD(c 2.7mM,MeOH),λ max (Δε)295(-1.13),265(+2.73),245(-2.58),230(-0.51),215(-17.66)nm.

[0063] Compound 13: yellow oil; [α] 2 D 0 -26 (c 0.03, MeOH); 1 H and 13 C NMR data are shown in Table 5; HR-ESI-MS (m / z): 412.1745 [M+Na] + (calcd for C 21 H 27 NO6Na,412.1736); ECD(c 2.6mM,MeOH),λ max (Δε)295(-2.10),266(+1.69),246(-2.88),228(+0.85),215(-18.21)nm.

[0064] Compound 14: yellow oil; [α] 2 D 0 -38 (c 0.03, MeOH); 1 H and 13C NMR data are shown in Table 5; HR-ESI-MS (m / z): 390.1919 [M+H] + (calcd for C 21 H 28 NO6,390.1917); ECD(c 2.6mM,MeOH),λ max (Δε)294(-2.43),264(+3.74),245(-2.30),229(+1.97),213(-36.86)nm.

[0065] Compound 15: white powder; [α] 2 D 0 -29 (c 0.03, MeOH); 1 H and 13 C NMR data are shown in Table 5; HR-ESI-MS (m / z): 404.2067 [M+H] + (calcd for C 22 H 30 NO6,404.2073); ECD(c 2.5mM,MeOH),λ max (Δε)296(-1.52),264(+3.23),245(-2.60),230(-0.44),215(-18.14)nm.

[0066] Compound 16: white powder; [α] 2 D 0 -16 (c 0.07, MeOH); 1 H and 13 C NMR data are shown in Table 6; HR-ESI-MS (m / z): 444.2083 [M+Na] + (calcd for C 26 H 29 NO4Na,444.2151); ECD(c 20mM,MeOH),λ max (Δε)281(-0.70),265(-0.48),247(-0.83),227(+0.05),215(-0.03)nm.

[0067] Compound 17: white powder; [α] 2 D 0 -31.5 (c 0.07, MeOH); 1 H and 13C NMR data are shown in Table 6; HR-ESI-MS (m / z): 378.1876 [M+Na] + (calcd for C 22 H 27 NO3Na,378.1883); ECD(c 25mM,MeOH),λ max (Δε)260(-2.04),248(-1.45),221(-3.09),211(-6.96)nm.

[0068] Table 1. Compounds 1, 2, and 3 1 H and 13 C NMR data

[0069]

[0070] Table 2. Compounds 4, 5, and 6 1 H and 13 C NMR data

[0071]

[0072] Table 3. Compounds 7, 8, and 9 1 H and 13 C NMR data

[0073]

[0074]

[0075] Table 4. Compounds 9, 10, and 11 1 H and 13 C NMR data

[0076]

[0077] Table 5. Compounds 12, 13, 14, and 15 1 H and 13 C NMR data

[0078]

[0079]

[0080] Table 6. Compounds 16 and 17 1 H and 13 C NMR data

[0081]

[0082]

[0083] Example 2: Application of pyridopyrone alkaloid compounds in protecting APAP-induced acute liver injury

[0084] 1. Culture of Mouse Hepatocyte AML-12 Cells

[0085] AML-12 cells were cultured in a 10 cm cell culture dish with complete culture medium in a 37°C, 5% CO2 incubator. The cell culture medium was changed every other day. The specific steps were as follows: remove the AML-12 cell culture dish from the incubator, discard the old culture medium in the dish; rinse with 2 mL of PBS and discard; add 7 mL of complete culture medium; and return to the incubator for culture.

[0086] 2. MTT method to screen the activity of each component in the process of separation of crude extract from single fermentation

[0087] a. AML-12 cell culture and experimental grouping: The cell culture experimental steps were the same as above; the experiment was divided into a blank control group, an APAP model group, and a fermented crude extract administration group according to differences in cell treatment.

[0088] b. APAP drug preparation: Accurately weigh 0.4548 g APAP, add 30 mL PBS to fully dissolve, and filter through a 0.22 μm sterilizing filter to obtain a 100 mM APAP stock solution for use.

[0089] c. AML-12 cells were treated with APAP for 12 h to simulate the in vivo ALI environment: 100 mM APAP solution was diluted 10 times with complete culture medium to prepare 10 mM APAP working solution; the crude extract was separated and combined by silica gel column chromatography to obtain multiple components, 2 mg of each component was accurately weighed, 10 μL DMSO was added to fully dissolve, and a 200 mg / mL drug stock solution was prepared, and the drug stock solution was diluted with APAP working solution to a 100 mg / mL working solution for later use.

[0090] d. MTT method to detect AML-12 cell injury: AML-12 cells were seeded in 96-well plates, and complete culture medium, 10mM APAP working solution, and 10mM APAP working solution containing extracts were added, 100μL / well, 5% CO2, 37℃ for 24h, and the experimental groups were control group, model group, and extract administration group (n=5). After 24h, the medium containing drugs was discarded, and 100μL complete culture medium and 20μL MTT solution (5mg / mL, i.e. 0.5% MTT) were added to each well, and the culture was continued for 4h; after 4h, the culture was terminated, and the culture medium in the well was carefully aspirated; 150μL dimethyl sulfoxide was added to each well, and the well was placed on a shaker for 10min at a low speed to fully dissolve the crystals. The absorbance value of each well at 490nm was detected by an enzyme marker.

[0091] 3. MTT assay to detect the effects of compounds 1-7 and compounds 9-16 on the survival rate of APAP-induced AML-12 cells

[0092] Weigh 1 mg of each compound accurately, add appropriate volume of DMSO to dissolve according to molecular weight, add 10mM APAP working solution to prepare compound concentration gradient of 50, 25, 10, 5μM working solution for standby use. MTT method to detect AML-12 cell injury: AML-12 cells were inoculated in 96-well plates, and complete culture medium, 10mM APAP working solution, and 10mM APAP working solution containing different concentrations of each compound were added, 100μL / well, 5% CO2, 37℃ culture for 24h, and the experimental groups were control group, model group, and compound administration group (n=5). After 24h, the medium containing the drug was aspirated and discarded, and 100μL complete culture medium and 20μL MTT solution (5mg / mL, i.e. 0.5% MTT) were added to each well, and the culture was continued for 4h; after 4h, the culture was terminated, and the culture medium in the well was carefully aspirated; 150μL dimethyl sulfoxide was added to each well, and the plate was placed on a shaker and shaken at low speed for 10min to fully dissolve the crystals. The absorbance of each well was detected by an enzyme-labeled instrument at 490 nm.

[0093] 4. Detection of alanine aminotransferase (ALT) in the supernatant of APAP-induced AML-12 cells by compounds 1-7, 9-10 and 12-15

[0094] Weigh 1 mg of each compound accurately, add an appropriate volume of DMSO to dissolve according to the molecular weight, and add 10mM APAP working solution to prepare a working solution with a compound concentration of 5μM for standby use. ALT detection of each compound in the supernatant of AML-12 cells: ① Treatment and experimental grouping of AML-12 cells: The cell culture experimental steps are the same as above. The cells are inoculated in a 24-well plate, and complete culture medium, 10mM APAP working solution, and working solution containing 5μM compounds are added, 100μL / well; ② According to the differences in cell treatment, they are divided into blank control group, APAP model group, and drug administration experimental group. Collect the supernatant of AML-12 cell culture medium in 24-well plates. ③ Prepare a standard curve using sodium pyruvate standard solutions of different concentrations. ④ Add 20μL of ALT matrix solution to the measurement wells, control wells, and standard wells, and add 5μL of the sample to be tested to the control wells. After mixing, react in a 37℃ incubator for 30min. ⑤ Add different concentrations of sodium pyruvate standard solution to the standard wells; add 20 μL of 2,4-dinitrotoluene hydrazine solution to each well, add 5 μL of the sample to be tested to the control wells, mix well, and react in a 37°C incubator for 20 minutes. ⑥ Add 200 μL of 0.4 mol / L sodium hydroxide solution to each well, gently shake the 96-well plate, leave it at room temperature for 15 minutes, and use a full-wavelength microplate reader to measure the absorbance value of each well at a wavelength of 510 nm.

[0095] 5. KEGG and GO analysis

[0096] The mechanism of compound 12 in improving acute liver injury (ALI) was further investigated using network pharmacology and structural virtual screening methods, and 105 targets were identified through the SwissTargetPrediction database. Subsequently, a search of the OMIM and Genecards online databases generated 8547 targets related to ALI. The intersection of these data visualized in the Venny diagram revealed 95 possible targets for compound 12 to improve ALI ( Figure 3 The GO biological functions and KEGG pathway enrichment analysis of these common targets were analyzed using the clusterProfiler package in R (v.4.3.2). The GO analysis results showed that most of the identified target proteins were related to various protein kinases, such as 3-kinase / protein kinase B, protein kinase complex, serine / threonine protein kinase, protein serine kinase, protein tyrosine kinase, and heptad repeat kinase ( Figure 4 ). In addition, KEGG results showed that most of these targets were enriched in the PI3K-Akt signaling pathway, Rap1 signaling pathway, FOXO signaling pathway, and EGFR signaling pathway ( Figure 5 At the same time, the protein-protein interaction (PPI) network was constructed using the STRING database and visualized using Cytoscape software, and 10 core targets (PIK3CA, MTOR, GRB2, BCL2, HSP90AA1, STAT3, EGFR, SRC, TNF, and AKT1) were identified. Figure 6 ). Molecular docking was performed to explore the interaction between compound 12 and the selected targets. The binding energy and molecular docking of compound 12 with each target protein are shown in Figure 7 and Figure 8 The lowest docking energy (-8.35 kcal / mol) was observed with the target EGFR protein. The analysis revealed the interaction between compound 12 and EGFR, indicating that compound 12 improves acute liver injury by regulating downstream cascade reactions such as ras / raf / MEK / MAPK, PI3K / Akt and STAT pathways.

[0097] 6. Experimental Results

[0098] The cell survival rates of normal mouse liver cells AML-12, APAP-induced liver injury AML-12 cells, and APAP-induced liver injury cells treated with different concentrations (5, 10, 25, 50 μM) of compounds 1-7 and 9-16 are shown in the table. Figure 1. The changes in cell viability at 5μM and 10μM dosing concentrations were expressed as cell protection rates, and calculated to obtain Table 7. The cell survival rate experiment showed that compounds 1-7, 9-10 and 12-15 significantly improved the cell survival rate of APAP-induced liver injury cells. Alanine aminotransferase (ALT) is an enzyme localized in hepatocytes and released into the blood during liver damage. It is a specific marker for acute lung injury. In order to determine the cytoprotective activity of the bioactive compounds, the ALT content in the cell supernatant was further tested under the administration of compounds 1-7, 9-10 and 12-15. The results are shown in Table 7. Figure 2 , and expressed as the reduction rate of ALT level, calculated to obtain Table 8. Compounds 1-7, 9-10 and 12-15 can significantly reduce the ALT level in the supernatant of APAP-induced ALI when administered at a concentration of 5 μM or 10 μM, showing ALI protective activity. In addition, bioinformatics analysis showed that these pyranopyridones may improve ALI by regulating downstream cascade reactions involving pathways such as ras / raf / MEK / MAPK, PI3K / Akt and STAT. The structural diversity of this type of pyranopyridone alkaloids provides valuable chemical precursors for the development of ALI protective drugs.

[0099] Table 7. Cell protection rate of compounds 1-7 and compounds 9-16 against APAP-induced liver injury cells

[0100]

[0101] Table 8. Reduction rate of ALT level in APAP-induced AML-12 cells by compounds 1-7, 9-10 and 12-15

[0102]

[0103]

[0104] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A pyranopyridone alkaloid compound, characterized in that: Any one of compounds 1-17 shown in the following structural formula: The structural formula of compounds 1, 2, 3, and 4: The group combinations of compounds 1, 2, 3, and 4 are shown in the following table: The structural formulas of compounds 5, 6, 7, 8, 9, 10 and 11 are: The group combinations of compounds 5, 6, 7, 8, 9, 10, and 11 are shown in the following table: The structural formulas of compounds 12, 13, 14 and 15 are: The group combinations of compounds 12, 13, 14, and 15 are shown in the following table: The structural formula of compound 16: The structural formula of compound 17:

2. The method for preparing the pyranopyridone alkaloid compound according to claim 1, characterized in that: The following steps are involved: Aspergillus aculeatinus WHUF0198 is inoculated alone or simultaneously with Penicillium sp. DM27 into a liquid culture medium for separate fermentation or co-culture fermentation, and the pyranopyridone alkaloid compound is separated from the fermentation product.

3. The method for preparing a pyranopyridone alkaloid compound according to claim 2, characterized in that: The following steps are involved: Aspergillus aculeatinus WHUF0198 is inoculated alone or simultaneously with Penicillium sp.DM27 into a liquid culture medium for fermentation alone or co-culture, the fermentation liquid and mycelium are separated, the mycelium is crushed and ultrasonically extracted with acetone, and the recovered acetone is concentrated under reduced pressure and then combined with the fermentation liquid; The combined solution is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract; or the combined solution is added to a macroporous resin for adsorption, and the macroporous resin is subjected to column chromatography using a methanol / water gradient elution to obtain components containing different methanol contents, and the component containing the pyridinepyrone alkaloid is determined, and these components are combined and concentrated under reduced pressure to obtain a crude extract; The crude extract is separated by silica gel column chromatography to obtain smaller components, which are then separated by gel column chromatography. The characteristic peaks of pyranopyridone alkaloid compounds are purified by semi-preparative liquid phase to obtain pyranopyridone alkaloid compounds.

4. Use of the pyranopyridone alkaloid compound according to claim 1 in the preparation of a medicament for treating acute liver injury.

5. The use according to claim 4, characterized in that: The pyranopyridone alkaloid compound is one or more of compounds 1-17 in claim 1.

6. The use according to claim 4, characterized in that: The acute liver injury is drug-induced liver injury.

7. The use according to claim 4, characterized in that: The acute liver injury is acetaminophen-induced acute liver injury.

8. A drug for treating acute liver injury, characterized in that: Contains the pyranopyridone alkaloid compound according to claim 1.

9. The drug according to claim 8, characterized in that: Contains one or more of the compounds 1-17 in claim 1.

10. The drug according to claim 8 or 9, characterized in that: Also contains pharmaceutically acceptable excipients.

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