2-pyrone compound as well as preparation method and application thereof

Abstract

The invention discloses a 2-pyrone compound as well as a preparation method and application of the 2-pyrone compound, and the 2-pyrone compound is hispidin (1), hispolone (2) and 3, 3 '-methene-bis [6-(3, 4-dihydroxyphenyl)-4-hydroxyl-2H-pyran-2-one] (MBP) (3). The preparation method comprises the following steps: fermenting Inonotus hispidus, and separating and purifying a fermented product through silica gel column chromatography and a high performance liquid chromatography (HPLC) method to obtain compounds 1-3. Pharmacological experiments show that the 2-pyrone compound disclosed by the invention has the activity of promoting angiogenesis of zebra fish, and can be applied to research on medicines for preventing and treating cardiovascular diseases. A new thought is provided for searching medicines with angiogenesis promoting activity and used for treating or preventing cardiovascular diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a 2-pyrone compound and a preparation method and application thereof. Background Art

[0002] According to statistics from the World Health Organization, more than 18 million people die from cardiovascular disease each year, ranking first among all causes of death. In my country, 4.58 million people die from cardiovascular disease each year, which is equivalent to 2 in every 5 adults suffering from cardiovascular disease. Cardiovascular disease is a disease of the heart and blood vessels, including arrhythmia, atherosclerosis, cardiomyopathy, congenital heart disease, heart failure, etc., which has a great impact on the quality of life of patients and is a common disease that seriously threatens human life and health. In recent years, the number of people suffering from cardiovascular disease has been increasing year by year. Unfortunately, there are only a few expensive drugs that can be used to treat the disease, so it is of great significance to explore new cardiovascular drugs.

[0003] Zebrafish is another important model organism after mice. The advantages of zebrafish, such as small size, low breeding cost, transparent body, and easy genetic manipulation, make it an ideal model organism. Since Professor George Streisinger of the University of Oregon in the United States used zebrafish for research on genetics and developmental biology in the 1970s, zebrafish has been increasingly used in the study of human diseases. In recent years, zebrafish has been widely used as a model organism in the field of cardiovascular diseases such as human arrhythmia and drug screening and evaluation. Against the above background, the focus of this patent is that the inventor obtained three compounds with significant angiogenesis-promoting activity in zebrafish, hispidin, hispolon, and MBP, through separation and identification in the continuous research on Inonotus hispidus. There is no report on the angiogenesis-promoting activity of the compound in zebrafish, and there is no related drug on the market. Summary of the invention

[0004] The present invention aims to provide three 2-pyrone compounds with angiogenesis-promoting activity, namely hispidin, hispolon and MBP, as well as preparation methods and new uses thereof, so as to provide new ideas for the research on drugs with angiogenesis-promoting activity for treating or preventing cardiovascular diseases.

[0005] The technical solutions adopted to achieve the above purpose are as follows:

[0006] A 2-pyrone compound selected from the group consisting of hispidin (1), hispolon (2) and 3,3'-methylene-bis[6-(3,4-dihydroxystyryl)-4-hydroxy-2H-pyran-2-one](MBP) (3); the structural formula of the compound is:

[0007]

[0008] The compound is separated and purified from the fermentation product of Inonotus hispidus.

[0009] The application of the 2-pyrone compounds in the preparation of angiogenesis-promoting drugs.

[0010] The use of the 2-pyrone compounds in the preparation of angiogenesis-promoting drugs is characterized in that the compounds 1-3 show angiogenesis-promoting activity on zebrafish in a dose-dependent manner.

[0011] The use of the 2-pyrone compound in the preparation of angiogenesis-promoting drugs is characterized in that the effective concentration of the compound 3 for exerting angiogenesis-promoting activity is 10-50 μmol / mL.

[0012] The application of the 2-pyrone compounds in the preparation of drugs for treating or preventing cardiovascular diseases.

[0013] A pharmaceutical composition comprises the 2-pyrone compounds 1-3, and optionally contains a pharmaceutically acceptable carrier.

[0014] The pharmaceutical composition comprises compounds 1-3 and a pharmaceutically acceptable carrier. The pharmaceutical composition contains 0.1-99% of the compounds 1-3 by mass, and the rest is a pharmaceutical carrier or excipient.

[0015] A pharmaceutical composition, characterized in that the dosage form of the drug is any one of tablets, capsules, and granules.

[0016] The preparation process of the 2-pyrone compound comprises the steps of fermenting Phellinus hispidus and extracting the fermented product: preparing a recovery solid culture medium according to a weight ratio of water: glucose: potato: agar = 100:2:20:2, and adjusting the pH value thereof to 6.5; inoculating a certain amount of Phellinus hispidus onto the recovery solid culture medium, and culturing the culture medium at a constant temperature of 28°C in an incubator for 3 days; preparing a liquid culture medium according to a weight ratio of water: corn starch: glucose: peptone: potassium dihydrogen phosphate: magnesium sulfate = 100:1:1:0.2:0.1:0.05, and adjusting the pH value thereof to 6.5; taking Phellinus hispidus on the recovery solid culture medium, and then extracting the fermented product; preparing a liquid culture medium according to a weight ratio of water: corn starch: glucose: peptone: potassium dihydrogen phosphate: magnesium sulfate = 100:1:1:0.2:0.1:0.05, and adjusting the pH value thereof to 6.5; extracting the ... and then extracting the fermented product; and then extracting the fermented product; and then extracting the fermented product; and then extracting the fermented product; and then extracting the fermented product; and finally extracting the fermented product; and finally extracting the fermented product; and finally extracting the fermented product; and finally extracting the fermented product; and finally extracting the fermented product; and finally extracting the fermented product; and finally extracting the fermented product; and finally extract hispidus, inoculating it into a bottle containing the above-mentioned liquid culture medium, and conducting a constant temperature static culture at 25°C for 30 days; the liquid culture medium was placed in a fermentation barrel, and an equal volume of ethyl acetate was added, and the extraction was static for 3 days. After 4 extractions, the extracts were combined and the ethyl acetate was recovered in a rotary evaporator to obtain a crude extract; the obtained crude extract was separated by column chromatography to obtain compound 1-3; wherein the chromatographic separation method includes silica gel column chromatography, gel column chromatography, and semi-preparative high performance liquid chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is compound 1 of the present invention 1 H-NMR spectrum;

[0018] Figure 2 It is compound 1 of the present invention 13 C-NMR spectrum;

[0019] Figure 3 It is compound 2 of the present invention 1 H-NMR spectrum;

[0020] Figure 4 It is compound 2 of the present invention 13 C-NMR spectrum;

[0021] Figure 5 It is compound 3 of the present invention 1 H-NMR spectrum;

[0022] Figure 6 It is compound 3 of the present invention 13 C-NMR spectrum;

[0023] Figure 7 It is a fluorescence microscope image of the angiogenesis-promoting activity test of compounds 1-3 in the present invention in zebrafish;

[0024] Figure 8 These are the experimental results of the test on the angiogenesis-promoting activity of compounds 1-3 in the present invention in zebrafish. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0026] Example 1: Preparation of Compound 1-3

[0027] A solid medium for recovery was prepared according to a weight ratio of water: glucose: potato: agar = 100:2:20:2, and its pH value was adjusted to 6.5; a certain amount of Inonotus hispidus was inoculated onto the above solid medium for recovery, and cultured in a 28°C incubator for 3 days; a liquid culture solution was prepared according to a weight ratio of water: corn starch: glucose: peptone: potassium dihydrogen phosphate: magnesium sulfate = 100:1:1:0.2:0.1:0.05, and its pH value was adjusted to 6.5; Inonotus hispidus on the solid medium for recovery was taken. hispidus, inoculated it into a triangular flask (230 bottles) filled with 300 mL of the above liquid culture solution, and cultured at 25°C for 30 days; after 30 days, pour the culture medium after the fermentation into a fermentation barrel, add a small amount of ethyl acetate to inactivate it, use a wall breaking machine to obtain tissue homogenate, add an equal volume of ethyl acetate, and perform water bath ultrasound for 30 minutes. After 4 extractions, the extracts are combined and the ethyl acetate is recovered in a rotary evaporator to obtain a crude extract (about 33 g).

[0028] The crude extract was dissolved in methanol and chromatographed on a 100-200 mesh silica gel column, and then eluted three times with petroleum ether-ethyl acetate (1:0, 50:1, 40:1, 20:1, 10:1, 8:1, 5:1, 2:1, 1:1, 1:1.5 and 0:1) and dichloromethane-methanol (50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:1.5 and 0:1). The eluents in the 1:1.5 and 0:1 sections of petroleum ether:ethyl acetate were collected and concentrated to a solid state under reduced pressure to obtain a crude product; the crude product was then eluted with a polarity decreasing gradient by a rapid liquid phase preparation system, and the absorption peaks analyzed by the liquid phase spectrum were combined into 31 components (Fr.1-Fr.31). The component Fr.18 was dissolved in methanol, eluted with acetonitrile-water at a flow rate of 4 mL / min and a volume ratio of 20:80, and separated by high performance liquid chromatography HPLC to prepare the compound hispidin; the component Fr.16 was dissolved in methanol, eluted with acetonitrile-water at a flow rate of 4 mL / min and a volume ratio of 40:60, and separated by high performance liquid chromatography HPLC to prepare the compounds hispolon and MBP.

[0029] Example 2: Structural Identification of Compounds 1-3

[0030] The structures of the compounds hispidin, hispolon, and MBP were determined through comprehensive analysis of high-resolution mass spectrometry, ultraviolet spectroscopy, nuclear magnetic resonance, and other data. Their physicochemical properties are as follows:

[0031] Compound 1: hispidin, yellow amorphous powder, positive ion mass spectrum ESI-MS m / z given at 246.05 [M+H] + Quasi-molecular ion peak, according to 1 H and 13 C NMR indicates that the molecular formula of compound 1 is C 13 H 10 O5, unsaturation degree is 9, UV (MeCN) λmax 209 (8.78), 219 (8.84), 238 (8.77), 251 (8.79), 284 (8.84) and 371 (8.96) nm; its H NMR spectrum and C NMR spectrum data are shown in Table 1.

[0032] Table 1. H NMR and C NMR data of compound 1

[0033]

[0034] Compound 2: hispolon, white powder, positive ion mass spectrum ESI-MS m / z is 220.07 [M+H] + Quasi-molecular ion peak, 1 H and 13 C NMR showed that the molecular formula of compound 2 was C 12 H 12 O4, unsaturation is 7, UV (MeCN) λmax 230 (8.70), 259 (8.76), 276 (8.78) and 371 (8.91) nm; its H NMR spectrum and C NMR spectrum data are shown in Table 2.

[0035] Table 2. H NMR and C NMR data of compound 2

[0036]

[0037]

[0038] Compound 3: MBP, yellow powder, positive ion mass spectrum ESI-MS m / z is 505.1 [M+H] + Quasi-molecular ion peak, 1 H and 13 C NMR showed that the molecular formula of compound 3 was C 27 H 20 O 10, the unsaturation degree is 18, UV(MeCN)λmax 210(5.28),218(5.32),240(5.06),255(5.11),288(4.57) and 389(5.50)nm; its hydrogen NMR spectrum and carbon NMR spectrum data are shown in Table 3.

[0039] Table 3. H NMR and C NMR data of compound 3

[0040]

[0041] Example 3

[0042] In order to further verify the beneficial effects of the compounds of the present invention, the compounds 1-3 were tested for their angiogenesis-promoting activity in zebrafish. The specific experiment is as follows:

[0043] 1. Test methods:

[0044] 1. Obtaining zebrafish embryos: Male and female zebrafish were fed separately at 28°C, 14h light / 10h dark conditions to ensure normal spawning. Healthy and mature male and female zebrafish were placed in a mating tank at a ratio of 1:1. The next morning, fertilized eggs were obtained. After disinfection and washing of the fertilized eggs, they were transferred to zebrafish embryo culture water (5.0mM NaCl, 0.17mMKCl, 0.4mM CaCl2 and 0.16mM MgSO4) and cultured at 28°C with controlled light. Each sample was dissolved into different concentrations, and PTK787 and samples of different doses were added to a 24-well plate.

[0045] 2. Angiogenesis activity test: The positive drug is ferulic acid (100 μg / mL), and 10 zebrafish embryos that have developed to 24 hpf and have been decapped are added to each well (two replicate wells are set for each sample group). The zebrafish are added with PTK787 (final concentration 0.25 μg / mL) to create a zebrafish vascular injury model. After mixing, cover and place in a constant temperature incubator. After 24 hours of drug administration, observe and photograph the intersegmental blood vessel growth under a fluorescence microscope and count the number of intersegmental blood vessels.

[0046] 2. Test results

[0047] The experimental results show that the number of intersegmental blood vessels in the zebrafish PTK787 injury model group is significantly different from that in the experimental group (## indicates p≤0.01 compared with the control group; ** indicates p≤0.01 compared with PTK787; p is significant). Compounds 1-3 have angiogenic activity in zebrafish induced by PTK787, and as the concentration of the compound increases, the angiogenic activity in zebrafish is more obvious. In addition, compound 3 has a stronger angiogenic effect in zebrafish than the positive drug ferulic acid at a concentration of 20 μmol / mL. (See attached for details.) Figure 7 and 8 )

[0048] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A 2-pyrone compound selected from the group consisting of hispidin (1), hispolon (2) and 3,3'-methylene-bis[6-(3,4-dihydroxystyryl)-4-hydroxy-2H-pyran-2-one](MBP) (3); the structural formula of which is:

2. The 2-pyrone compound according to claim 1, characterized in that The compound is separated and purified from the fermentation product of Inonotus hispidus.

3. Use of the 2-pyrone compound described in claim 1 in the preparation of angiogenesis-promoting drugs.

4. The use of the 2-pyrone compound according to claim 3 in the preparation of angiogenesis-promoting drugs, characterized in that: The compounds 1-3 showed pro-angiogenic activity in zebrafish in a dose-dependent manner.

5. The use of the 2-pyrone compound according to claim 3 in the preparation of angiogenesis-promoting drugs, characterized in that: The effective concentration of the compound 3 for promoting angiogenesis is 10-50 μmol / mL.

6. Use of the 2-pyrone compound according to claim 1 in the preparation of a drug for treating or preventing cardiovascular diseases.

7. A pharmaceutical composition, characterized in that The 2-pyrone compounds 1-3 according to claim 1 optionally contain a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, comprising compounds 1-3 and a pharmaceutically acceptable carrier, wherein the mass fraction of the pharmaceutical composition is 0.1-99% of compounds 1-3, and the rest is a pharmaceutically acceptable carrier or excipient.

9. A pharmaceutical composition according to claim 7, characterized in that: The dosage form of the medicine is any one of tablets, capsules and granules.

10. The 2-pyrone compound according to claim 1, characterized in that: The method comprises the following steps of fermenting Phellinus hispidus and extracting the fermented product: preparing a recovery solid culture medium according to a weight ratio of water: glucose: potato: agar = 100:2:20:2, and adjusting the pH value thereof to 6.5; inoculating a certain amount of Phellinus hispidus onto the recovery solid culture medium, and culturing the culture medium at a constant temperature of 28°C in an incubator for 3 days; preparing a liquid culture medium according to a weight ratio of water: corn starch: glucose: peptone: potassium dihydrogen phosphate: magnesium sulfate = 100:1:1:0.2:0.1:0.05, and adjusting the pH value thereof to 6.5; taking the Phellinus hispidus on the recovery solid culture medium, and then adding the liquid culture medium to ... hispidus, inoculating it into a bottle containing the above-mentioned liquid culture medium, and conducting a constant temperature static culture at 25°C for 30 days; the liquid culture medium was placed in a fermentation barrel, and an equal volume of ethyl acetate was added, and the extraction was static for 3 days. After 4 extractions, the extracts were combined and the ethyl acetate was recovered in a rotary evaporator to obtain a crude extract; the obtained crude extract was separated by column chromatography to obtain compound 1-3; wherein the chromatographic separation method includes silica gel column chromatography, gel column chromatography, and semi-preparative high performance liquid chromatography.

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