Quinolinone compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of extraction and separation, in particular to a quinolinone compound as well as a preparation method and application thereof. The quinolinone compound provided by the invention can participate in the reaction of cells to nitrogen compounds / organic nitrogen compounds / organic ring compounds, and biological processes of body fluid level regulation, protein phosphorylation, peptidyl amino acid modification, MAPK cascade regulation and the like; the compound has the biological functions of kinase activity, phosphotransferase activity, transcription factor binding, protein structural domain specific binding, ubiquitin-like protein ligase binding and the like, can be applied to preparation of drugs for treating diabetes mellitus, and has a treatment value on diabetes mellitus; moreover, the quinolinone compound provided by the invention can also be applied to preparation of medicines for treating fatty liver, cervical cancer, lung diseases or Hodgkin's lymphoma, and has anti-inflammatory and anti-tumor dual activities.

Description

Technical Field

[0001] The invention relates to the technical field of extraction and separation, and in particular to a quinolinone compound and a preparation method and application thereof. Background Art

[0002] Phyllostachys nigra var.henonis is a plant of the genus Phyllostachys under the subfamily Bambusoideae. It is a variant of Phyllostachys nigra. Its culms are purple-black, about 7 to 18 meters tall, with thick culm walls up to 5 mm. There are very few dark brown spots on the top of the sheath. Phyllostachys nigra is one of the medicinal bamboo species, and its culms can be used as medicine. Phyllostachys nigra culms are also the raw materials for preparing traditional Chinese medicine bamboo juice and bamboo shavings, which have the effects of expectoration and cough relief.

[0003] Modern pharmacological studies have shown that the extracts of Phyllostachys pubescens have anti-inflammatory, anti-cancer, cholesterol-lowering, melanin-inhibiting and neuroprotective effects. The rich secondary metabolites in Phyllostachys pubescens provide the material basis for its diverse pharmacological activities. At present, the chemical components isolated from Phyllostachys pubescens are mostly known compounds such as syringol, catechol and phthalic acid, and no quinolinone compounds that can be used to prepare drugs for the treatment of diabetes have been found. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a quinolinone compound and a preparation method and application thereof. The quinolinone compound provided by the present invention can be used to prepare a drug for treating diabetes.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a quinolinone compound having a structure shown in Formula 1:

[0007]

[0008] The present invention provides a method for preparing the quinolinone compound described in the above technical solution, comprising the following steps:

[0009] The plant of Phyllostachys genus is mixed with a lower alcohol extractant, and then concentrated after extraction to obtain an alcohol extract; the lower alcohol extractant is a lower alcohol or a lower alcohol aqueous solution, and the volume fraction of the lower alcohol in the lower alcohol extractant is 70-100%;

[0010] The alcohol extract is dissolved and resuspended in water, and the obtained resuspended solution is extracted with a water-saturated n-butanol solution and then concentrated to obtain an n-butanol extract;

[0011] The n-butanol extract is subjected to separation by an adsorption resin chromatographic column and then concentrated to obtain a separation component by the adsorption resin chromatographic column; the eluent used for separation by the adsorption resin chromatographic column comprises ethanol-water, and the volume fraction of ethanol in the ethanol-water is 0 to 20%;

[0012] The components separated by the adsorption resin chromatographic column are subjected to medium-pressure preparative chromatographic separation to obtain component A3; the eluent used in the medium-pressure preparative chromatographic separation includes an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 5 to 100%;

[0013] The A3 component is separated by a gel chromatography column and then concentrated to obtain an A3i component; the eluent used in the gel chromatography column separation includes water;

[0014] The A3i component is separated by a semi-preparative chromatography column to obtain quinolinone compounds; the eluent used in the semi-preparative chromatography column separation includes a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 12-15%.

[0015] Preferably, the lower alcohol includes methanol and / or ethanol; the extraction is performed 3 to 5 times;

[0016] The ratio of the mass of the Phyllostachys genus plant to the volume of the low-grade alcohol extractant used for a single extraction is 1 g: 3-5 mL;

[0017] The extraction temperature is 10-35° C., and the single extraction time is 20-30 hours.

[0018] Preferably, the extraction is performed 3 to 5 times; and the volume ratio of the resuspension to the n-butanol solution saturated with water for single extraction is 1:1 to 3.

[0019] Preferably, the adsorption resin used in the adsorption resin chromatographic column includes HP-20;

[0020] The elution mode of the adsorption resin chromatographic column separation is gradient elution. During the gradient elution, the volume fractions of ethanol in ethanol-water are 0% and 20% respectively.

[0021] Preferably, the chromatographic column filler used in the medium pressure preparative chromatographic separation comprises C 18 Bonded silica gel;

[0022] The elution mode of the medium-pressure preparative chromatographic separation is gradient elution. During the gradient elution, the volume fraction of ethanol in the ethanol aqueous solution is 5%, 10%, 15%, 20%, 25%, 30%, 50% and 100% in sequence.

[0023] Preferably, the chromatographic column used for the gel chromatography column separation comprises a Sephadex LH-20 gel chromatography column.

[0024] Preferably, the chromatographic column used for the semi-preparative chromatographic column separation comprises C 18 Semi-preparative columns.

[0025] Preferably, the extraction comprises a first extraction and a second extraction performed sequentially;

[0026] The volume fraction of the lower alcohol in the lower alcohol extractant used in the first extraction is 90-100%;

[0027] The volume fraction of the lower alcohol in the lower alcohol extractant used in the second extraction is 70-80%.

[0028] The present invention provides the use of the quinolinone compounds described in the above technical solution in preparing medicines for treating diabetes, fatty liver, cervical cancer, lung disease or Hodgkin's lymphoma.

[0029] The present invention provides a quinolinone compound having a structure shown in Formula 1. The quinolinone compound provided by the present invention can participate in biological processes such as cell response to nitrogen compounds / organic nitrogen compounds / organic ring compounds, regulation of body fluid levels, protein phosphorylation, peptide amino acid modification, MAPK cascade regulation, etc., has biological functions such as kinase activity, phosphotransferase activity, transcription factor binding, protein domain specific binding, ubiquitin-like protein ligase binding, etc., can be used in the preparation of drugs for treating diabetes, and has therapeutic value for diabetes; moreover, the quinolinone compound provided by the present invention has dual anti-inflammatory and anti-tumor activities, and can also be used in the preparation of drugs for treating fatty liver, cervical cancer, lung disease or Hodgkin's lymphoma.

[0030]

[0031] The preparation method of the quinolinone compounds provided by the invention is simple to operate, and the chromatographic column separation only needs to be eluted with water and / or lower alcohol, which has low cost, small environmental pollution and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a high-resolution mass spectrum of quinolinone compounds;

[0033] Figure 2 The hydrogen spectrum of quinolinone compounds (DMSO-d 6 );

[0034] Figure 3 The carbon spectrum of quinolinone compounds (DMSO-d 6 );

[0035] Figure 4 DEPT135 spectrum of quinolinone compounds (DMSO-d 6 )

[0036] Figure 5 HSQC spectrum of quinolinone compounds (DMSO-d 6 );

[0037] Figure 6 The HMBC spectrum of quinolinone compounds (DMSO-d 6 );

[0038] Figure 7 This is the target protein interaction network diagram of quinolinone compounds;

[0039] Figure 8 It is the key target map of the predicted target protein interaction network of quinolinone compounds;

[0040] Fig. 9 This is the key subnetwork diagram of predicted targets of quinolinone compounds;

[0041] Fig.10 This is a bar graph showing the results of gene ontology enrichment analysis of predicted targets of quinolinone compounds;

[0042] Fig.11 This is a bar graph showing the enrichment analysis results of Kyoto Encyclopedia of Genes and Genomes for the predicted targets of quinolinone compounds;

[0043] Fig.12 This is a bar chart showing the enrichment analysis results of the DisGeNET database for predicted targets of quinolinone compounds. DETAILED DESCRIPTION

[0044] The present invention provides a quinolinone compound having a structure shown in Formula 1:

[0045]

[0046] The quinolinone compound provided by the present invention is systematically named as quinolinone-3-O-glucose (quinolin-4(1H)-one-3-O-β-D-glucopyranoside), and its molecular formula is C 15 H 17 NO 7 .

[0047] The present invention provides a method for preparing the quinolinone compound described in the above technical solution, comprising the following steps:

[0048] The plant of Phyllostachys genus is mixed with a lower alcohol extractant, and then concentrated after extraction to obtain an alcohol extract; the lower alcohol extractant is a lower alcohol or a lower alcohol aqueous solution, and the volume fraction of the lower alcohol in the lower alcohol extractant is 70-100%;

[0049] The alcohol extract is dissolved and resuspended in water, and the obtained resuspended solution is extracted with a water-saturated n-butanol solution and then concentrated to obtain an n-butanol extract;

[0050] The n-butanol extract is subjected to separation by an adsorption resin chromatographic column and then concentrated to obtain a separation component by the adsorption resin chromatographic column; the eluent used for separation by the adsorption resin chromatographic column comprises ethanol-water, and the volume fraction of ethanol in the ethanol-water is 0 to 20%;

[0051] The components separated by the adsorption resin chromatographic column are subjected to medium-pressure preparative chromatographic separation and then concentrated to obtain component A3; the eluent used in the medium-pressure preparative chromatographic separation includes an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 5 to 100%;

[0052] The A3 component is separated by a gel chromatography column and then concentrated to obtain an A3i component; the eluent used in the gel chromatography column separation includes water;

[0053] After the A3i component is dissolved in water, it is separated by a semi-preparative chromatography column to obtain quinolinone compounds; the eluent used in the semi-preparative chromatography column separation includes a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 13%.

[0054] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0055] The invention mixes Phyllostachys genus plants and a low-grade alcohol extractant, extracts and then concentrates (referred to as the first concentration) to obtain an alcohol extract.

[0056] In the present invention, the plant of the genus Phyllostachys preferably includes Phyllostachys pubescens. In a specific embodiment, the plant of the genus Phyllostachys genus can be one or more of Phyllostachys pubescens stalks, Phyllostachys pubescens leaves and Phyllostachys pubescens whips; the moisture content of the plant of the genus Phyllostachys genus is preferably 30-50%. In a specific embodiment, the moisture content of the plant of the genus Phyllostachys genus can be 30%, 35%, 40%, 45% or 50%; the plant of the genus Phyllostachys genus is preferably further crushed before use; the particle size of the crushed plant of the genus Phyllostachys genus is preferably 20-60 meshes. In a specific embodiment, the particle size of the crushed plant of the genus Phyllostachys genus can be 20 mesh, 25 mesh, 30 mesh, 35 mesh, 40 mesh, 45 mesh, 50 mesh, 55 mesh or 60 mesh.

[0057] In the present invention, the volume fraction of the lower alcohol in the lower alcohol extractant is 70-100%. In a specific embodiment, the volume fraction of the lower alcohol in the lower alcohol extractant can be 70%, 75%, 80%, 85%, 90% or 100%. The lower alcohol preferably includes methanol and / or ethanol. The number of extractions is preferably 3-5 times. In a specific embodiment, the number of extractions can be 3, 4 or 5 times. The ratio of the mass of the Phyllostachys genus plant to the volume of the lower alcohol extractant for a single extraction is preferably 1 g: 3-5 mL. In a specific embodiment, the Phyllostachys genus plant The ratio of the mass of the substance to the volume of the low-grade alcohol extractant for a single extraction can be 1g:3mL, 1g:3.5mL, 1g:4mL, 1g:4.5mL or 1g:5mL; the extraction temperature is preferably 10-35°C. In a specific embodiment, the extraction temperature can be 10°C, 15°C, 20°C, 25°C or 30°C; the single extraction time is preferably 20-30h. In a specific embodiment, the single extraction time can be 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h or 30h. In the present invention, the extraction preferably includes a first extraction and a second extraction performed sequentially; the volume fraction of the lower alcohol in the lower alcohol extractant used in the first extraction is preferably 90-100%, and in a specific embodiment, the volume fraction of the lower alcohol in the lower alcohol extractant used in the first extraction can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98% or 100%; the number of the first extraction is preferably 1-2 times; the volume fraction of the lower alcohol in the lower alcohol extractant used in the second extraction is preferably 70-80%, and in a specific embodiment, the volume fraction of the lower alcohol in the lower alcohol extractant used in the second extraction can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%; the number of the second extraction is preferably 2-3 times

[0058] After the extraction is completed, the present invention preferably further comprises a first concentration of the obtained extract. In the present invention, the temperature of the first concentration is preferably 40-50°C. In a specific embodiment, the temperature of the first concentration can be 42°C, 44°C, 45°C, 47°C, 49°C or 50°C; the first concentration preferably comprises reduced pressure concentration; the present invention has no special requirements for the pressure of the reduced pressure concentration, and the pressure of the reduced pressure concentration conventionally used by those skilled in the art can be used. The present invention has no special limitation on the time of the first concentration, as long as the alcohol extract can be concentrated to a constant weight.

[0059] After obtaining the alcohol extract, the present invention dissolves the alcohol extract in water and resuspends it, extracts the resuspended liquid with a water-saturated n-butanol solution and then concentrates it (referred to as the second concentration) to obtain the n-butanol extract.

[0060] In the present invention, the ratio of the mass of the alcohol extract to the volume of water is preferably 1g:3-5mL. In a specific embodiment, the ratio of the mass of the alcohol extract to the volume of water can be 1g:3mL, 1g:3.5mL, 1g:4mL, 1g:4.5mL or 1g:5mL. In the present invention, the temperature of the extraction is preferably 10-35°C. In a specific embodiment, the temperature of the extraction can be 10°C, 15°C, 20°C, 25°C, 30°C or 35°C; the number of extractions is preferably 4 times; the time of a single extraction is preferably 20-30min. In a specific embodiment, the time of a single extraction can be 20min, 22min, 25min, 26min, 28min or 30min; the volume ratio of the alcohol extract to the n-butanol solution saturated with water for a single extraction is preferably 1:1-3. In a specific embodiment, the volume ratio of the alcohol extract to the n-butanol solution saturated with water for a single extraction can be 1:1, 1:1.5, 1:2, 1:2.5 or 1:3.

[0061] After the extraction is completed, the present invention preferably further comprises a second concentration of the obtained organic phase, and the obtained concentrate is dried to obtain a n-butanol extract. In the present invention, the conditions of the second concentration are preferably the same as those of the first concentration, and are not repeated here. The present invention has no special restrictions on the time of the second concentration, and the organic phase can be concentrated to a constant weight. In the present invention, the drying is preferably freeze drying; the freeze drying pressure is preferably 0 to 10Pa, and in a specific embodiment, the freeze drying pressure can be 0Pa, 2Pa, 4Pa, 5Pa, 6Pa, 8Pa or 10Pa; the freeze drying temperature is preferably -70 to -80°C, and in a specific embodiment, the freeze drying temperature can be -70°C, -72°C, -74°C, -76°C, -78°C or -80°C. The present invention has no special restrictions on the freeze drying time, and the concentrate can be completely dried.

[0062] After obtaining the n-butanol extract, the present invention separates the n-butanol extract using an adsorption resin chromatographic column and then concentrates the extract (referred to as the third concentration) to obtain a separation component using an adsorption resin chromatographic column.

[0063] In the present invention, the n-butanol extract is dissolved by adding water before being separated by an adsorption resin chromatography column; the present invention has no special limitation on the amount of the dissolving water, as long as the n-butanol extract can be completely dissolved.

[0064] In the present invention, the adsorption resin used in the adsorption resin chromatographic column preferably includes HP-20; the eluent used in the adsorption resin chromatographic column separation includes ethanol-water, the volume fraction of ethanol in the ethanol-water is 0-20%, in a specific embodiment, the volume fraction of ethanol in the ethanol-water can be 0%, 5%, 10%, 15% or 20%; the amount of the ethanol-water is preferably 3-5BV (column volume), in a specific embodiment, the amount of the ethanol-water can be 3BV, 3.5BV, 4BV, 4.5BV or 5BV. In the present invention, during the separation process of the adsorption resin chromatographic column, the volume fraction of ethanol in the ethanol-water is preferably 0% and 20% in sequence, the amount of ethanol-water with a volume fraction of 0% is preferably 2BV; the amount of ethanol-water with a volume fraction of 20% is preferably 3BV. The adsorption resin chromatographic column separation components obtained by eluting with ethanol-water with a volume fraction of 20% are subsequently treated.

[0065] After the separation by the adsorption resin chromatographic column is completed, the present invention preferably further comprises a third concentration of the obtained 20% ethanol eluate to obtain an adsorption resin chromatographic column separation component. In the present invention, the conditions of the third concentration are preferably the same as those of the first concentration, and are not repeated here. The present invention has no special limitation on the time of the third concentration, as long as the adsorption resin chromatographic column separation component can be concentrated to a constant weight.

[0066] After obtaining the components separated by the adsorption resin chromatographic column, the present invention performs medium-pressure preparative chromatographic separation on the components separated by the adsorption resin chromatographic column to obtain component A3.

[0067] In the present invention, the components separated by the adsorption resin chromatographic column are further dissolved by adding ethanol before the medium-pressure preparative chromatographic separation; the present invention has no special limitation on the amount of ethanol used for dissolution, as long as the components separated by the adsorption resin chromatographic column can be completely dissolved.

[0068] In the present invention, the chromatographic column filler used in the medium pressure preparative chromatographic separation includes C 18Bonded silica gel, in a specific embodiment, the chromatographic column filler used in the medium-pressure preparative chromatography separation can be ODS-A (wherein A refers to the chromatographic column model); the chromatographic column system used in the medium-pressure preparative chromatography separation preferably includes a Sepacore X10 chromatography system; the medium-pressure preparative chromatography separation preferably includes the following steps: after adsorbing the A3 component using a chromatographic column filler, separating it using a SepacoreX10 chromatography system. In the present invention, the eluent used in the medium-pressure preparative chromatographic separation includes an ethanol aqueous solution, the volume fraction of ethanol in the ethanol aqueous solution is 5-100%, and in a specific embodiment, the volume fraction of ethanol in the ethanol aqueous solution can be 5%, 10%, 15%, 20%, 25%, 30%, 50%, 70%, 80% or 100%; during the medium-pressure preparative chromatographic separation process, the volume fraction of ethanol in the ethanol aqueous solution is preferably 5%, 10%, 15%, 20%, 25%, 30%, 50% and 100% in sequence, and the amount of the ethanol aqueous solution with a single ethanol volume fraction is preferably 2-3BV. In a specific embodiment, the amount of the ethanol aqueous solution with a single ethanol volume fraction can be 2BV, 2.2BV, 2.4BV, 2.6BV, 2.8BV or 3BV. In the present invention, the medium-pressure preparative chromatographic separation process preferably also includes high-performance liquid chromatography detection, which is detected at a wavelength of 254nm, with each 0.1-0.3BV (in a specific embodiment, it can be 0.1BV, 0.2BV or 0.3BV) as a fraction, and the same fractions are combined to obtain 10 components, which are successively recorded as A1 component, A2 component, A3 component, A4 component, A5 component, A6 component, A7 component, A8 component, A9 component and A10 component, and the A3 component is subsequently processed. In the present invention, the detector used for detection at a wavelength of 254nm preferably includes a photodiode array detector (PDAD).

[0069] After obtaining the A3 component, the present invention separates the A3 component by gel chromatography column and then concentrates it (recorded as the fifth concentration) to obtain the A3i component.

[0070] In the present invention, the A3 component is preferably first concentrated (referred to as the fourth concentration) and dissolved in ethanol, and then separated by medium-pressure preparative chromatography. In the present invention, the conditions for the fourth concentration are preferably the same as those for the first concentration, which will not be described in detail. The present invention does not specifically limit the time for the fourth concentration, as long as the A3 component can be concentrated to a constant weight.

[0071] In the present invention, the volume ratio of the A3 component to the gel used for gel chromatography column separation is preferably 1:90-110. In a specific embodiment, the volume ratio of the A3 component to the gel used for gel chromatography column separation can be 1:90, 1:95, 1:100, 1:105 or 1:110. Before the A3 component is separated by gel chromatography column, it is preferred that water is added for dissolution. The present invention has no special limitation on the amount of water used for dissolution, as long as it can completely dissolve the A3 component.

[0072] In the present invention, the chromatographic column used for the gel chromatography column separation preferably includes a Sephadex LH-20 gel chromatography column; the eluent used for the gel chromatography column separation includes water; during the gel chromatography column separation process, it is preferably also included to perform high performance liquid chromatography detection, with each 0.02 to 0.05BV (in a specific embodiment, it can be 0.02BV, 0.03BV, 0.04BV or 0.05BV) as a fraction, and the same fractions are combined to obtain 19 components, which are sequentially recorded as A3a component, A3b component, A3c component, A3d component, A3e component, A3f component, A3g component, A3h component, A3i component, A3j component, A3k component, A3l component, A3m component, A3n component, A3o component, A3p component, A3q component, A3r component and A3s component, and the A3i component is subjected to subsequent treatment.

[0073] After obtaining the A3i component, the present invention separates the A3i component using a semi-preparative chromatography column (Shimadzu) to obtain quinolinone compounds.

[0074] The present invention preferably concentrates the A3i component in sequence (referred to as the fifth concentration), dries and dissolves in water, and then separates it by semi-preparative chromatography. In the present invention, the conditions for the fifth concentration are preferably the same as those for the first concentration, which will not be described in detail here. The present invention does not have any special restrictions on the time of the fifth concentration, as long as the A3i component can be concentrated to a constant weight. In the present invention, the drying is preferably freeze-drying. In the present invention, the conditions for the freeze-drying are preferably the same as those described above, which will not be described in detail here. The present invention does not have any special restrictions on the amount of water used for dissolving, as long as the dried A3i component can be completely dissolved.

[0075] In the present invention, the chromatographic column used for the semi-preparative chromatographic column separation preferably comprises C 18 Semi-preparative chromatographic column; the C 18The chromatographic column filler of the semi-preparative chromatographic column preferably includes ODS-AQ; the eluent used for separation by the semi-preparative chromatographic column includes a methanol-water solution, the volume fraction of methanol in the methanol-water solution is 12-15%, and in a specific embodiment, the volume fraction of methanol in the methanol-water solution can be 12%, 13%, 14% or 15%; the elution method used for separation by the semi-preparative chromatographic column is preferably isocratic elution; the isocratic elution process also includes ultraviolet light detection of the eluted components obtained by the isocratic elution; the detection wavelength of the ultraviolet light detection is preferably 239nm.

[0076] The present invention provides the use of the quinolinone compounds described in the above technical scheme in the preparation of drugs for treating diabetes, fatty liver, cervical cancer, lung disease or Hodgkin's lymphoma. The quinolinone compounds provided by the present invention can participate in biological processes such as cell response to nitrogen compounds / organic nitrogen compounds / organic ring compounds, regulation of body fluid levels, protein phosphorylation, peptide amino acid modification, MAPK cascade regulation, etc., have biological functions such as kinase activity, phosphotransferase activity, transcription factor binding, protein domain specific binding, ubiquitin-like protein ligase binding, etc., can be used in the preparation of drugs for treating diabetes, and have therapeutic value for diabetes; moreover, the quinolinone compounds provided by the present invention have dual anti-inflammatory and anti-tumor activities, and can also be used in the preparation of drugs for treating fatty liver, cervical cancer, lung disease or Hodgkin's lymphoma.

[0077] In order to further illustrate the present invention, a quinolinone compound provided by the present invention and its preparation method and application are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0078] Example 1

[0079] Take fresh bamboo stalks of Phyllostachys pubescens, remove the leaves, cut into bamboo pieces with a width of less than 3 cm, and crush them with a hammer mill to obtain bamboo stalk powder with a particle size of 20 mesh. Take 53 kg of bamboo stalk powder, first extract it once with a 95% ethanol aqueous solution at 25°C, filter it, and then extract the residue twice with a 95% ethanol aqueous solution. After filtering, the filtrate is concentrated under reduced pressure at 45°C to obtain 2246 g of alcohol extract. The extraction time is 24 hours each time, and the extraction process is fully stirred every 6 hours. The ratio of the mass of bamboo stalk powder to the volume of ethanol aqueous solution used for a single extraction is 1g:4mL.

[0080] The obtained alcohol extract was dissolved and resuspended with distilled water to obtain a solution, and the obtained solution was extracted 4 times with a water-saturated n-butanol solution. The obtained organic phase was concentrated under reduced pressure at 45°C to recover n-butanol, and freeze-dried to obtain 446 g of n-butanol extract, wherein the volume ratio of the alcohol extract to the water-saturated n-butanol solution for a single extraction was 1:1.

[0081] The obtained n-butanol extract was dissolved and resuspended in distilled water, and separated by adsorption resin chromatography column using macroporous resin HP-20. 2BV of distilled water and 3BV of 20% ethanol aqueous solution were used as mobile phases for elution. The eluent with 20% ethanol aqueous solution was concentrated under reduced pressure at 45°C to obtain 61g of separation components by adsorption resin chromatography column.

[0082] The components were separated by an adsorption resin column obtained by dissolving in ethanol, and C 18 Bonded silica gel filler ODS-A (12nmS-50μm) was used for adsorption, and after the solvent was evaporated and removed, a dry column was used for separation by a medium pressure preparative chromatography system Sepacore X10. The elution was carried out in sequence with 5%, 10%, 15%, 20%, 25%, 30%, 50%, and 100% ethanol aqueous solutions. During the elution process, the detection was carried out at 254nm. When no chromatographic peak appeared, the mobile phase of the next concentration was replaced; each 0.2BV was a fraction, and after detection by a photodiode array detector (PDAD, a detector of HPLC), the same fractions were combined to obtain 10 components, which were successively recorded as A1 component, A2 component, A3 component, A4 component, A5 component, A6 component, A7 component, A8 component, A9 component, and A10 component. The A3 component was concentrated under reduced pressure at 45°C to obtain 10.2g of dry A3 component.

[0083] The dried A3 component was dissolved in distilled water and separated by Sephadex LH-20 gel chromatography column, with the sample loading amount being 1% of the volume of the gel column. Distilled water was used as the eluent, and each 0.05 BV was a fraction. The same fractions were combined after PDAD detection to obtain 19 fractions in total, which were successively recorded as A3a component, A3b component, A3c component, A3d component, A3e component, A3f component, A3g component, A3h component, A3i component, A3j component, A3k component, A3l component, A3m component, A3n component, A3o component, A3p component, A3q component, A3r component and A3s component. The A3i component was concentrated under reduced pressure at 45°C and freeze-dried to obtain 1210.2 mg of dried A3i component.

[0084] The obtained dried A3i component was dissolved in 10 mL of distilled water and separated by semi-preparative liquid chromatography. The chromatographic column was ODS-AQ (250×20 mm; 12 nm). Isocratic elution was performed with a methanol aqueous solution with a volume fraction of 13%. The detection wavelength was 239 nm. Repeated accumulation was performed to obtain 6.3 mg of quinolinone compounds, wherein the flow rate of the isocratic elution was 1 mL / min, the full wavelength was (200-400 nm), and the retention time was 14.5 min.

[0085] The structural formula (including the reference number) of the quinolinone compound prepared in this example is shown in Formula 2; the NMR data (DMSO-d6) of the quinolinone compound is shown in Table 1.

[0086]

[0087] Table 1 NMR data of quinolinone compounds (DMSO-d6)

[0088]

[0089]

[0090] Among them, the UV maximum absorption wavelength and the molecular weight obtained by high-resolution mass spectrometry of the quinolinone compounds are as follows: UV (MeOH) λmax (logε) 239.2nm; HRESIMS m / z: 322.0929 [MH]-, (calculated for 322.0932).

[0091] Figure 1 This is a high-resolution mass spectrum of quinolinone compounds; Figure 2 The hydrogen spectrum of quinolinone compounds (DMSO-d 6 ); Figure 3 The carbon spectrum of quinolinone compounds (DMSO-d 6 ).Depend on Figure 1 to Figure 3 As shown in Table 1, high-resolution mass spectrometry and nuclear magnetic resonance data show that the molecular formula of the prepared compound is C 15 H 17 O 7N, and the degree of unsaturation is 8 or 9. The H NMR spectrum showed that the compound contained five aromatic ring hydrogen signals, namely δH 8.18 (dd, 8.3, 1.4 Hz, 1H), δH 8.09 (s, 1H), δH 7.66 (ddd, 8.3, 6.6, 1.4 Hz, 1H), δH 7.62 (dd, 8.2, 1.4 Hz, 1H) and δH 7.34 (ddd, 8.3, 6.6, 1.4 Hz, 1H). Among them, the integral constant of the δH 8.18 peak group is about one hydrogen atom, and the coupling constants of this peak group are 8.3Hz and 1.4Hz, respectively, representing 3J and 4J coupling, indicating that this hydrogen atom has only one side with adjacent hydrogen atoms; the integral of the δH 7.66 peak group corresponds to one hydrogen atom, and the coupling constants of this peak group are 8.3Hz, 6.6Hz, and 1.4Hz, respectively, representing 3J, 3J and 4J coupling, indicating that this hydrogen atom has adjacent hydrogen atoms on both sides, and the peak shape analysis of the δH 7.62 and δH 7.34 peak groups is the same as above. In summary, the four hydrogen atoms corresponding to the δH 8.18, δH 7.66, δH 7.62 and δH 7.34 peak groups are connected together to form an AA'BB' spin coupling system, suggesting that there are two adjacent substituents on the aromatic ring (A) of this compound. The δH 8.09 peak group corresponds to a hydrogen atom, and the peak shape is a single peak, indicating that there is another aromatic ring (B), and there is only one hydrogen atom on the aromatic ring (B). δH4.63 (d, 7.9Hz, 1H) is the anomeric hydrogen signal, and the coupling constant is 7.9, indicating that the compound contains a D-glucose connected by a β-glycosidic bond. From this, it can be further determined that the compound does not contain a nitro group (-NO2), so the degree of unsaturation is 8. The C NMR spectrum shows that the compound contains a carbonyl signal δC 171.7, eight aromatic ring carbon signals δC 139.2, δC 138.1, δC 133.5, δC131.3, δC 125.5, δC 125.0, δC 123.0, δC119.0; combined with the DEPT spectrum, it can be seen that δC 139.2, δC 138.1, δC 125.5 are quaternary carbons. The signals of the β-D-glucose unit are δC 103.8 (anomeric carbon), δC 77.4, δC 76.3, δC 73.0, δC69.9, δC 61.2. Considering the molecular formula of the compound, it can be further inferred that δC 133.5 (C-2) and δC 139.2 (C-9) are two nitrogen-containing carbon atoms. The HMBC spectrum showed that H-1' (δH 4.63) was correlated with C-3 (δC 138.1), proving that the β-D-glucose unit was connected to C-3. In addition, the key HMBC-related signals H-1' / C-3, H-5 / C-4, H-2 / C-4, and H-2 / C-9 suggested that the compound was a quinolinone compound.

[0092] Example 2

[0093] Virtual screening of biological activities of quinolinone compounds

[0094] The reverse virtual screening technology based on pharmacophore model and structure was used to predict the biological activity of quinolinone compounds. The 3D structural model of quinolinone compounds was constructed using Chemdraw20.0, and the energy was minimized by MM2 force field. The target prediction and screening were performed by PharmMapper (z score>0.8), SEA (z score>10), Super-Pred (probability>60%; Model accuracy>80%), NetInfer (score>0.8) and SwissTargetPrediction database, respectively. All database parameters selected the default options to generate the target information of quinolinone compounds. The results of the five databases were merged, and the duplicate targets were removed to obtain the predicted biological activity targets of quinolinone compounds. The information of the top 100 targets is shown in Table 2.

[0095] Table 2 Predicted biological activity targets of quinolinone compounds (TOP100)

[0096]

[0097]

[0098] The protein-protein interaction (PPI) network was predicted using the STRING database. The species was set to Homo sapiens to obtain protein interaction information. The topological index was calculated using Cytoscape (V3.7.2). The protein-protein interaction network was constructed based on the degree. The results are shown in Figure 2. Figure 7 The top 15 key targets were screened by MCC algorithm, and the results are shown in Table 3 and Figure 8 As shown; and the MCODE plug-in was used to calculate the key sub-networks including betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), and the method based on local average connectivity (LAC). The results are shown in Table 4 and Fig. 9 As shown. Figure 7 to Figure 9As shown in Tables 3 and 4, GAPDH is the most critical target among the key targets, with an MCC value of 1.89E+09. In the key subnetwork, HIF1A is the core target and the center of the network. GAPDH is also in a core position in the subnetwork. By merging the key target set with the targets in the key subnetwork and removing duplicates, the core targets are as follows: HIF1A, GSK3B, CCND1, IL6, GAPDH, CDK1, CCNB1, CDK2, ABL1, HRAS, LYN, MCL1, CCNA2, CCNA1, NFE2L2, AR, EGFR, NFKB1, MMP9, and MAPK1.

[0099] Table 3 Key targets of quinolinone compound target protein interaction network

[0100]

[0101]

[0102] Table 4 Key subnetworks of the quinolinone compound target protein interaction network

[0103] Target Points Node Status BC CC DC EC LAC NC HIF1A 13.77 Seed 523.27 0.64 15.00 0.21 19.10 45.03 GSK3B 13.77 Clustered 361.90 0.62 14.00 0.18 17.06 30.21 CCND1 13.77 Clustered 194.95 0.61 15.00 0.20 20.50 36.75 IL6 13.77 Clustered 1503.48 0.73 14.00 0.23 18.30 58.37 GAPDH 13.77 Clustered 2849.25 0.81 15.00 0.27 18.88 79.04 CDK1 12.99 Clustered 89.70 0.56 14.00 0.13 16.06 20.42 CCNB1 12.99 Clustered 24.73 0.54 12.00 0.11 16.33 18.68 CDK2 12.69 Clustered 202.83 0.59 14.00 0.15 16.11 24.23 ABL1 11.98 Clustered 132.72 0.57 11.00 0.13 15.40 19.06 HRAS 11.82 Clustered 58.46 0.56 11.00 0.15 17.56 22.17 LYN 11.74 Clustered 65.31 0.56 9.00 0.12 14.21 17.33 MCL1 11.63 Clustered 69.01 0.56 14.00 0.14 16.44 20.89 CCNA2 11.56 Clustered 54.48 0.56 11.00 0.11 15.38 18.57 CCNA1 11.43 Clustered 7.58 0.51 10.00 0.09 13.78 15.03 NFE2L2 11.43 Clustered 28.62 0.53 9.00 0.11 13.00 14.44 AR 11.36 Clustered 36.34 0.54 12.00 0.12 15.92 18.85

[0104] The predicted targets and core targets were analyzed by gene ontology (GO) using Metascape v3.5.20230501 database. Fig.10 ), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the results are as follows Fig.11 As shown; the predicted targets were enriched based on the DisGeNET database, and the results are shown Fig.12 As shown in the figure, GO entries and KEGG pathways with p-value < 0.01 were considered significantly enriched. The GO enrichment results and KEGG enrichment results of predicted targets and core targets were intersected to obtain the most critical biological activity information of quinolinone compounds. Fig.11 and Fig.12It can be seen that quinolinone compounds are mainly involved in biological processes such as cell response to nitrogen compounds / organic nitrogen compounds / organic ring compounds, regulation of body fluid levels, protein phosphorylation, peptide amino acid modification, MAPK cascade regulation, etc., and have biological functions such as kinase activity, phosphotransferase activity, transcription factor binding, protein domain-specific binding, ubiquitin-like protein ligase binding, etc. The sites of action include dendrites, postsynapses, axons, cysts, receptor complexes, membrane rafts and other cellular components, and the pathways of action are cancer pathways (hsa05200), PI3K-Akt signaling pathway (hsa04151), bladder cancer (hsa05219), chemical carcinogen-receptor activation (hsa05207), prostate cancer (hsa05215), the role of AGE-RAGE signaling pathway in diabetic complications (hsa04933), Ras signaling pathway (hsa04014), Rap1 signaling pathway (hsa04015), central carbon metabolism in cancer (hsa05230), calcium signaling pathway (hsa04020), cell senescence (hsa04218), etc. The results of DisGeNET database enrichment analysis showed that quinolinone compounds may act on the following diseases: experimental diabetes, breast cancer stage IV, lung disease, neuralgia, pancreatic tumors, unipolar depression, mood disorders, abdominal aortic aneurysm, cognitive impairment, vascular diseases, etc.

[0105] In summary, the quinolinone compounds provided by the present invention can participate in the regulation of multiple signal transduction pathways, especially PI3K-Akt, Ras, Rap1 and other signal pathways, which are widely involved in cell proliferation, migration, survival and other processes. Therefore, the compounds may have the function of regulating cell activities. Multiple pathways affected by quinolinone compounds are closely related to the occurrence and development of tumors, such as cancer pathways, bladder cancer, prostate cancer, etc. At the same time, DisGeNET analysis also showed that it is related to a variety of solid tumors. Combined with its effects on proliferation-related pathways, it is inferred that the compound has potential anti-tumor activity; quinolinone compounds are involved in the regulation of pathways related to inflammatory responses such as MAPK, and enrichment analysis shows that it is related to a variety of metabolic-related diseases, indicating that it may have anti-inflammatory and metabolic regulatory activities; quinolinone compounds can affect the activity of ubiquitin ligases, which are involved in protein degradation, which may be related to anti-inflammatory and anti-tumor activities; quinolinone compounds act on sites such as dendrites and receptor complexes, which are key sites for signal transduction, which also supports the activity of the compound in regulating signal pathways; DisGeNET analysis results also show that quinolinone compounds may be related to nervous system diseases, providing clues for the compound to regulate the central nervous system; the compound is involved in the enrichment of disease pathways such as tumors and inflammation, suggesting its potential dual anti-tumor and anti-inflammatory activities. Based on the current target prediction and enrichment analysis, it can be inferred that quinolinone compounds have multiple biological activities such as hypoglycemic, anti-tumor, anti-inflammatory, metabolic regulation and central nervous system, which provides an important reference for the subsequent pharmacodynamic research of the compound.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quinolinone compound, It is characterized in that It has the structure shown in formula 1:

2. The method for preparing the quinolinone compound according to claim 1, It is characterized in that The following steps are involved: The plant of Phyllostachys genus is mixed with a lower alcohol extractant for extraction to obtain an alcohol extract; the lower alcohol extractant is a lower alcohol or a lower alcohol aqueous solution, and the volume fraction of the lower alcohol in the lower alcohol extractant is 70 to 100%; The alcohol extract is dissolved and resuspended in water, and the obtained resuspended solution is extracted with a water-saturated n-butanol solution and then concentrated to obtain an n-butanol extract; The n-butanol extract is subjected to separation by an adsorption resin chromatographic column and then concentrated to obtain a separation component by the adsorption resin chromatographic column; the eluent used for separation by the adsorption resin chromatographic column comprises ethanol-water, and the volume fraction of ethanol in the ethanol-water is 0 to 20%; The components separated by the adsorption resin chromatographic column are subjected to medium-pressure preparative chromatographic separation to obtain component A3; the eluent used in the medium-pressure preparative chromatographic separation includes an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 5 to 100%; The A3 component is separated by a gel chromatography column to obtain an A3i component; the eluent used in the gel chromatography column separation includes water; The A3i component is separated by a semi-preparative chromatography column to obtain quinolinone compounds; the eluent used in the semi-preparative chromatography column separation includes a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 12-15%.

3. The preparation method according to claim 2, It is characterized in that The lower alcohol includes methanol and / or ethanol; the extraction times are 3 to 5 times; The ratio of the mass of the Phyllostachys genus plant to the volume of the low-grade alcohol extractant used for a single extraction is 1 g: 3-5 mL; The extraction temperature is 10-35° C., and the single extraction time is 20-30 hours.

4. The preparation method according to claim 2, It is characterized in that The number of extractions is 3 to 5 times; The volume ratio of the resuspension to the n-butanol solution saturated with water for single extraction is 1:1-3.

5. The preparation method according to claim 2, It is characterized in that The adsorption resin used in the adsorption resin chromatographic column includes HP-20; The elution mode of the adsorption resin chromatographic column separation is gradient elution. During the gradient elution, the volume fractions of ethanol in ethanol-water are 0% and 20% respectively.

6. The preparation method according to claim 2, It is characterized in that The chromatographic column filler used in the medium pressure preparative chromatographic separation includes C 18 Bonded silica gel; The elution mode of the medium-pressure preparative chromatographic separation is gradient elution. During the gradient elution, the volume fraction of ethanol in the ethanol aqueous solution is 5%, 10%, 15%, 20%, 25%, 30%, 50% and 100% in sequence.

7. The preparation method according to claim 2, It is characterized in that The chromatographic column used in the gel chromatography column separation includes a Sephadex LH-20 gel chromatography column.

8. The preparation method according to claim 2, It is characterized in that The chromatographic column used for the semi-preparative chromatographic column separation includes C 18 Semi-preparative columns.

9. The preparation method according to claim 2 or 3, It is characterized in that The extraction includes a first extraction and a second extraction performed sequentially; The volume fraction of the lower alcohol in the lower alcohol extractant used in the first extraction is 90-100%; The volume fraction of the lower alcohol in the lower alcohol extractant used in the second extraction is 70-80%.

10. Use of the quinolinone compound according to claim 1 in the preparation of a medicament for treating diabetes, fatty liver, cervical cancer, lung disease or Hodgkin's lymphoma.

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