A pyrone compound and its extraction method and application

Abstract

The present application provides a pyrone compound, which is extracted from fermentation liquor of plant endophytic fungi and has the effects of reducing blood sugar and reducing uric acid.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and specifically to a pyranone compound. Background Technology

[0002] Diabetes mellitus (DM) is a chronic disease caused by carbohydrate metabolism disorders. Type 2 diabetes (T2DM), also known as non-insulin-dependent diabetes mellitus, accounts for more than 90% of diabetes cases, causing immense suffering to patients. Alpha-glucosidase inhibitors are currently the main treatment for T2DM and play an important role in clinical application. However, only three alpha-glucosidase inhibitors have been approved by the FDA, all of which are natural products derived from microorganisms.

[0003] Uric acid (UA) is the final product of purine metabolism and is mainly excreted through the kidneys. Hyperuricemia (HUA) is a metabolic disease caused by purine metabolism disorders, primarily due to increased UA synthesis or decreased renal excretion, and is the biochemical basis of gout. Xanthine oxidase (XO) is a key enzyme in uric acid production and has become a good target for anti-hyperuricemia drugs. Clinical epidemiological data also show a close relationship between hyperuricemia and diabetes. In addition to its effects on diabetes itself, the influence of certain hypoglycemic drugs on UA ​​metabolism has attracted considerable attention in the medical community.

[0004] With economic development and improved living standards, the incidence of diabetes and hyperuricemia is increasing year by year, and is showing a trend towards affecting younger people. Currently, most clinically available hypoglycemic drugs have certain side effects. Therefore, it is crucial and holds great potential to explore novel natural products derived from microorganisms and develop drugs that simultaneously lower both blood sugar and uric acid. Summary of the Invention

[0005] To address the above technical problems, this invention provides a pyranone compound extracted from the fermentation broth of plant endophytic fungi, which has hypoglycemic and uric acid-lowering effects.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0007] A pyranone compound, characterized by the following structure:

[0008] .

[0009] The application of the pyranone compounds described in this invention in the preparation of hypoglycemic and uric acid-lowering products.

[0010] Preferably, the product includes a drug.

[0011] The extraction method for pyranone compounds of the present invention includes the following steps:

[0012] A. Fermentation: Ferment Diaporthe sp. CB10100 in rice or PDB medium to obtain fermentation product for later use;

[0013] B. Separation: The fermented product was extracted three times with ethyl acetate and then concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain the corresponding concentrates. The ethyl acetate fraction was separated by normal-phase silica gel column chromatography, ODS column chromatography gradient elution, and high-performance liquid chromatography to obtain the compound Ellagic acid C.

[0014] Preferably, the fungus Diaporthe sp. CB10100 is cultured first and then fermented.

[0015] More preferably, before fermentation, the fungus Diaporthe sp. CB10100 is cultured in a 1000 mL flat-bottomed conical flask containing 200 mL PBD solution and cultured at 30 ℃ and 200 rpm for 3 days to obtain the fungal seed fermentation broth. The fungal seed fermentation broth is inoculated at 10% of the volume of the large fermentation medium.

[0016] More preferably, each liter of culture medium used for fermentation comprises: 200 mL of a solution containing 35 g PDB / L in a 1000 mL Erlenmeyer flask, or 200 g of rice and 200 mL of water, sterilized for large-scale fermentation.

[0017] Preferably, in step B, the ethyl acetate fraction is subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase, and 10 fractions Fr.AJ are collected. Fraction Fr.B is eluted by methanol / water ODS column chromatography gradient to obtain 7 fractions Fr.B1-7. Fraction Fr.B6 is then separated by high performance liquid chromatography to obtain the compound Ellagic acid C.

[0018] More preferably, the volume ratio of the petroleum ether / ethyl acetate gradient elution varies to 20:1, 10:1, 1:1, 1:10, and 1:20.

[0019] The volume ratio changes of the methanol / water ODS gradient elution are: 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8;

[0020] The chromatographic conditions for high performance liquid chromatography are as follows:

[0021] Chromatographic column: Yuexu AQ-C18, 250 × 10 mm, 5 μm

[0022] Mobile phase: Acetonitrile (A): 0.2% formic acid water (B); Gradient elution was used, and the mobile phase ratio A:B was increased from 20:80 to 65:35 from 0 to 11 min, the mobile phase ratio was maintained at 65:35 from 11 to 12 min, the mobile phase ratio was changed from 65:35 to 20:80 from 12 to 19 min, and the mobile phase ratio was maintained at 20:80 from 19 to 22 min.

[0023] Flow rate: 3 mL / min;

[0024] Column temperature: 35 ℃. Attached Figure Description

[0025] Figure 1 It is the 1H NMR of the compound Ellagic acid C;

[0026] Figure 2 It is the 13C NMR of the compound Ellagic acid C;

[0027] Figure 3 It is DEPT90 of the compound Ellagic acid C;

[0028] Figure 4 It is DEPT135 of the compound Ellagic acid C;

[0029] Figure 5 This is the two-dimensional nuclear magnetic resonance spectrum (HSQC) of the compound Ellagic acid C;

[0030] Figure 6 It is the two-dimensional nuclear magnetic resonance spectrum (HMBC) of the compound Ellagic acid C;

[0031] Figure 7 This is the two-dimensional nuclear magnetic resonance spectrum (1H-1H COSY) of the compound Ellagic acid C;

[0032] Figure 8 This is a high-resolution mass spectrum of the compound Ellagic acid C. Detailed Implementation

[0033] To more clearly and in detail illustrate the objective and technical solution of this invention, the invention will be further described below through relevant embodiments. These embodiments are merely illustrative of the implementation methods of this invention and do not limit the scope of protection of this invention.

[0034] Example 1

[0035] A pyranone compound with the following structure:

[0036] .

[0037] Example 2

[0038] The extraction method for pyranone compounds of the present invention includes the following steps:

[0039] A. Fermentation: The fungus Diaporthe sp. CB10100 is fermented in rice to obtain fermented product for later use;

[0040] B. Separation: The fermentation product was extracted three times with ethyl acetate and concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain the corresponding concentrates. The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase, and 10 fractions Fr.AJ were collected. Fraction Fr.B was eluted with a methanol / water ODS column gradient to obtain 7 fractions Fr.B1-7. Fraction Fr.B6 was then separated by high performance liquid chromatography to obtain the compound Ellagic acid C.

[0041] Example 3

[0042] The extraction method for pyranone compounds of the present invention includes the following steps:

[0043] A. Fermentation: The fungus Diaporthe sp. CB10100 was fermented in PDB medium to obtain the fermentation product for later use;

[0044] Each liter of culture medium used for fermentation contains: 200 mL of a solution containing 35 g PDB / L added to a 1000 mL Erlenmeyer flask, sterilized, and used for large-scale fermentation.

[0045] B. Separation: The fermentation product was extracted three times with ethyl acetate and concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain the corresponding concentrates. The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase, and 10 fractions Fr.AJ were collected. Fraction Fr.B was eluted with a methanol / water ODS column gradient to obtain 7 fractions Fr.B1-7. Fraction Fr.B6 was then separated by high performance liquid chromatography to obtain the compound Ellagic acid C.

[0046] Example 4

[0047] The extraction method for pyranone compounds of the present invention includes the following steps:

[0048] A. Fermentation: Diaporthe sp. CB10100 fungus was first cultured in a 1000 mL flat-bottomed conical flask containing 200 mL PBD solution and cultured at 30 ℃ and 200 rpm for 3 days to obtain fungal seed fermentation broth. The fungal seed fermentation broth was inoculated at 10% of the volume of the large fermentation medium.

[0049] Each liter of culture medium used for fermentation contains: 200 g of rice and 200 mL of water added to a 1000 mL Erlenmeyer flask, sterilized, and then used for large-scale fermentation.

[0050] B. Separation: The fermentation product was extracted three times with ethyl acetate and concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain the corresponding concentrates. The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase, and 10 fractions Fr.AJ were collected. Fraction Fr.B was eluted with a methanol / water ODS column gradient to obtain 7 fractions Fr.B1-7. Fraction Fr.B6 was then separated by high performance liquid chromatography to obtain the compound Ellagic acid C.

[0051] The volume ratio of the petroleum ether / ethyl acetate gradient elution varied to 20:1, 10:1, 1:1, 1:10, and 1:20.

[0052] The volume ratio changes of the methanol / water ODS gradient elution are: 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8;

[0053] The chromatographic conditions for high performance liquid chromatography are as follows:

[0054] Chromatographic column: Yuexu AQ-C18, specifications: 250 × 10 mm, 5 μm

[0055] Mobile phase: Acetonitrile (A): 0.2% formic acid water (B); Gradient elution was used, and the mobile phase ratio A:B was increased from 20:80 to 65:35 from 0 to 11 min, the mobile phase ratio was maintained at 65:35 from 11 to 12 min, the mobile phase ratio was changed from 65:35 to 20:80 from 12 to 19 min, and the mobile phase ratio was maintained at 20:80 from 19 to 22 min.

[0056] Flow rate: 3 mL / min;

[0057] Column temperature: 35 ℃.

[0058] Structural analysis of Ellagic acid C:

[0059] Ellagic acid C underwent a complete structural characterization through comprehensive structural analysis.

[0060] (1) High-resolution mass spectrometry (HRMS) analysis yielded the [MH] of Ellagic acid C. - The molecular ion peak (m / z) is 303.0511, which corresponds to its standard molecular formula C. 15 H 13 O7 [C 15 H 12 O7-H] - The molecular weight values ​​are consistent with 303.0510. Figure 8 ).

[0061] (2) Ultraviolet spectroscopy analysis showed that Ellagic acid C contains three absorption peaks at 197.2 nm, 223.1 nm and 364.5 nm.

[0062] Ellagic acid C is pale yellow in color; UV (MeOH)max 197.2 nm, 223.1 nm, 364.5 nm are shown in [reference needed]. Figure 3 HRESIMS: Molecular formula C 15 H 12 Calculated value of O7: 303.0510, measured value: 303.0511, see [link / reference]. Figure 8 ;

[0063] (3) NMR data: see Table 1. Figure 1-4 It is a one-dimensional nuclear magnetic resonance spectrum. Figure 5-7 It is a two-dimensional nuclear magnetic resonance spectrum, in which Figure 1 It is Ellagic acid C. 1 H NMR, DMSO-d6 (400 MHz); Figure 2 It is Ellagic acid C. 13 C NMR, DMSO-d6 (100 MHz); Figure 3 It is DEPT90 of Ellagic acid C. Figure 4 It is DEPT135 of Ellagic acid C and Figure 5-7 It is a two-dimensional nuclear magnetic resonance spectrum, in which Figure 5 It is the HSQC of Ellagic acid C. Figure 6 It is HMBC of Ellagic acid C; Figure 7 It is Ellagic acid C. 1 H - 1 H COSY; Figure 8 This is a high-resolution mass spectrum of Ellagic acid C.

[0064]

[0065] Table 1

[0066]

[0067] In vitro inhibitory activity of compound Ellagic acid C against α-glucosidase

[0068] Glucosidase, a membrane-bound enzyme of the small intestinal epithelial cells, resides on the microvilli of the brush border cells and plays a crucial role in carbohydrate digestion. Therefore, it can serve as a drug target for the treatment of diabetes. Alpha-glucosidase inhibitors competitively inhibit the activity of alpha-glucosidase in the small intestine, delaying or inhibiting glucose absorption in the gut, thereby effectively reducing postprandial blood glucose spikes and regulating blood glucose levels. They play a vital role in suppressing postprandial hyperglycemia.

[0069] This invention describes the in vitro inhibitory activity assay for the compound Ellagic acid C. In an in vitro enzyme reaction system, the compound interacts with the enzyme by adding a substrate, enzyme, and buffer solution. The enzyme's UV absorbance level is then measured using a microplate reader, and its IC50 value is determined accordingly. 50 To determine the in vitro inhibitory activity of the target compound against α-glucosidase.

[0070] Experimental methods

[0071] Set up a 200 μL reaction system. Add 130 μL of PBS buffer to the sample group, followed by 10 μL of Ellagic acid C solution and 10 μL of 0.1 U / mL enzyme solution. Incubate at 37 °C for 20 min, then add 50 μL of 0.5 mM PNPG to each well, incubate at 37 °C for 40 min, and finally stop the reaction by adding 100 μL of Na2CO3 to each well.

[0072] Inhibition rate (%) =

[0073] In the formula, A s Indicates background group (Ellagic acid C+PBS), A t Indicates sample group (Ellagic acid C+ enzyme + PBS), A c This indicates the control group (enzyme + PBS) and A. b The blank group (PBS) used acarbose as a positive control.

[0074] Table 2. Reaction system for in vitro inhibitory activity experiment of α-glucosidase

[0075]

[0076] Table 3. In vitro inhibitory activity of Ellagic acid C and the positive control drug acarbose.

[0077]

[0078] As shown in Table 3, the IC50 of the compound Ellagic acid C on the inhibition of α-glucosidase is... 50 The IC50 value for acarbose is 356 ± 13.69 μM. 50 The concentration was 671.5 ± 0.17 μM, and the compound Ellagic acid C showed stronger activity than the positive control drug acarbose.

[0079] In vitro inhibitory activity of compound Ellagic acid C against xanthine oxidase

[0080] Xanthine oxidase (XO) is a flavoprotein protease with low specificity, widely distributed in organisms. It effectively catalyzes the conversion of xanthine or hypoxanthine into uric acid, making it a key enzyme in uric acid production. Xanthine oxidase inhibitors reduce uric acid production by inhibiting the activity of xanthine oxidase in the body, and have become a research hotspot in the relief and treatment of hyperuricemia. Currently, the main drugs used clinically to inhibit xanthine oxidase activity include allopurinol and febuxostat, which can cause a series of adverse reactions, such as liver dysfunction, allergic reactions, and acute renal failure. Therefore, xanthine oxidase inhibitors still have great research potential.

[0081] This invention describes the in vitro inhibitory activity assay for the compound ellagic acid C. The assay involves adding a substrate, enzyme, and buffer solution to an in vitro enzyme reaction system to allow the compound to interact with the enzyme. The UV absorbance level of the product uric acid is then measured using a microplate reader, and its IC50 value is subsequently determined. 50 To determine the in vitro inhibitory activity of the target compound against xanthine oxidase.

[0082] Experimental methods

[0083] To establish a 200 μL reaction system, 60 μL of sodium phosphate buffer solution was added to a 96-well microplate, along with 50 μL of samples of different concentrations. Then, 30 μL (or 5 U / L) xanthine oxidase (XO) solution preheated at 25 °C was added and mixed well. After incubation at 25 °C in the dark for 15 min, 60 μL of 150 μM xanthine substrate was added to initiate the reaction. After reacting at room temperature for 5 min, the absorbance was measured at 295 nm.

[0084] Inhibition rate (%) =

[0085] In the formula, As represents the sample group (Ellagic acid C + enzyme + PBS), As represents the control group (enzyme + PBS), Ab represents the blank sample group (Ellagic acid C + PBS), and A1 represents the blank control group (PBS) of the control group, with allopurinol as the positive control.

[0086] Table 4. In vitro inhibitory activity of Ellagic acid C and the positive control drug allopurinol

[0087]

[0088] As shown in Table 4, the IC50 of the compound Ellagic acid C on xanthine oxidase inhibition is... 50 The IC50 of allopurinol is 0.41 ± 0.09 μg / mL. 50 The concentration was 0.49 ± 0.06 μg / mL, indicating that the compound Ellagic acid C was more active than the positive control drug allopurinol.

[0089] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A pyranone compound, characterized in that, The structure is as follows: 。 2. The use of the pyranone compounds according to claim 1 in the preparation of hypoglycemic or hypouric acid-lowering drugs.

3. The extraction method for pyranone compounds according to claim 1, characterized in that, Includes the following steps: A. Fermentation: Ferment the fungus Diaporthe sp. CB10100 in rice or PDB medium to obtain the fermentation product for later use; B. Separation: The fermentation product was extracted three times with ethyl acetate and concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain the corresponding concentrates. The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase, and 10 fractions Fr.AJ were collected. Fraction Fr.B was eluted with a methanol / water ODS column gradient to obtain 7 fractions Fr.B1-7. Fraction Fr.B6 was then separated by high performance liquid chromatography to obtain pyranone compounds.

4. The extraction method for pyranone compounds according to claim 3, characterized in that, The fungus Diaporthesp. CB10100 was cultured first and then fermented.

5. The extraction method for pyranone compounds according to claim 4, characterized in that, Before fermentation, the fungus Diaporthe sp. CB10100 was cultured in a 1000 mL flat-bottomed conical flask containing 200 mL PDB solution at 30 °C and 200 rpm for 3 days to obtain the fungal seed fermentation broth. The fungal seed fermentation broth was inoculated at 10% of the volume of the fermentation medium.

6. The method for extracting pyranone compounds according to claim 5, characterized in that, The fermentation medium consists of 200 mL of a 35 g PDB / L solution or 200 g rice and 200 mL water added to a 1000 mL Erlenmeyer flask, sterilized, and then used for large-scale fermentation.

7. The extraction method for pyranone compounds according to claim 3, characterized in that, The volume ratio of the petroleum ether / ethyl acetate gradient elution varied to 20:1, 10:1, 1:1, 1:10, and 1:

20. The volume ratio changes of the methanol / water ODS column chromatography gradient elution are: 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8; The chromatographic conditions for high performance liquid chromatography are as follows: Column: Yuexu AQ-C18, 250×10mm, 5μm Mobile phase: Acetonitrile as phase A; 0.2% formic acid aqueous solution as phase B; Gradient elution was used, the mobile phase ratio A:B increased from 20:80 to 65:35 from 0-11 min, maintained at 65:35 from 11-12 min, changed from 65:35 to 20:80 from 12-19 min, and maintained at 20:80 from 19-22 min. Flow rate: 3 mL / min; Column temperature: 35℃.

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