Isoflavone compounds and preparation method and application thereof

Abstract

The present invention discloses an isoflavone compound, and its chemical formula is determined by nuclear magnetic resonance hydrogen spectrum, carbon spectrum, high-resolution mass spectrum and chemical method: After comparison, it is determined that the compound is a new isoflavone compound (compound 1), which is the first compound isolated from the traditional Chinese medicine hair fruit fish vine, and there is no report so far. At the same time, the inventor has also established a corresponding preparation method, extracting the rattan stem of hair fruit fish vine with ethanol solution, and then the extract is analyzed by high performance liquid chromatography, and then gradient elution is carried out using n-hexane ethyl acetate elution system, and further separated and prepared by reverse phase column chromatography and semi-preparative liquid chromatography. The lipid-lowering activity experiment proves that compound 1 has the effect of reducing the TG content of insulin-resistant HepG2 cells. Therefore, the isoflavone compound of the present invention has great development potential in the treatment and prevention of hyperlipidemia, high triglycerides.

Description

Technical Field

[0001] The invention belongs to the technical field of isoflavone compounds, and in particular relates to an isoflavone compound and a preparation method and application thereof. Background Art

[0002] Derris eriocarpa How is the dried vine and rhizome of Derris eriocarpa How, a plant of the genus Derris in the Fabaceae family. It is also known as "earth licorice" and "vine licorice". It is also called "vine sugar" in Zhuang language. It is a climbing shrub and is mainly distributed in Guangxi and Yunnan in my country. The resources of Derris eriocarpa How in Jingxi City, Guangxi are abundant and the sales volume is large. The rhizome of Derris eriocarpa How can be used as medicine. The stem is brownish yellow with protrusions on the surface. The cut surface is light yellow and fibrous. The texture is hard and not easy to break. Derris eriocarpa How has the effects of diuresis, dehumidification, antitussive and expectorant. As one of the components of the folk prescriptions of the "Zhuang nationality", it is used to treat cough and expectorant as well as chest tightness, fever, nephritis, cystitis and other diseases. Due to its significant therapeutic effect in folk medicine, it has attracted widespread attention from many scientific researchers.

[0003] The liver is the largest organ in the body and participates in many metabolic pathways of the body. In the process of lipid metabolism, it promotes fat digestion and absorption by secreting bile. It also participates in glucose uptake, synthesis, metabolism and storage. It is also the main target tissue of insulin resistance. Its metabolism directly affects glucose homeostasis and insulin sensitivity index. Therefore, liver cell damage or lipid metabolism disorder is particularly important for insulin resistance. Fatty acids are stored in cells in the form of TG. When the TG content is too high, it will cause fatty degeneration and then cause lipid metabolism disorder. Hyperlipidemia is one of the main risk factors for cardiovascular and cerebrovascular diseases such as coronary heart disease, diabetes, and atherosclerosis. Studies have shown that compounds in many natural products can regulate the liver lipid metabolism system to achieve the purpose of lowering blood lipids. Therefore, lipid-lowering active substances in natural products have become a research hotspot in the field of lipid-lowering.

[0004] At present, the research on the stem of Derris tricholoma includes thin layer chromatography, ultraviolet fingerprint identification, and pharmacological and pathological research such as anti-inflammatory, analgesic, cough and expectorant, but there are relatively few studies on its chemical composition and activity analysis, preparation technology, etc. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an isoflavone compound and a preparation method and application thereof, specifically a new isoflavone compound extracted and separated from the stem of Derris tricholoma and its application in lowering blood lipids.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] Isoflavone compounds, the chemical structure is:

[0008]

[0009] (Chemical name: 6-(2-hydroxy-3-methyl-3-enyl)-3-(4-hydroxyphenyl)-5,7-dimethoxyisoflavone).

[0010] The preparation method of the isoflavone compounds is to extract and separate the isoflavone compounds from the stems of Derris trichocarpa as raw materials.

[0011] The preparation method of the above-mentioned isoflavone compounds is to extract the stems of Derris trichocarpa with ethanol solution, and then the extract is analyzed by high performance liquid chromatography (HPLC), and then gradient elution is performed using a n-hexane-ethyl acetate elution system, and further separated and prepared by reverse phase column (ODS) chromatography and semi-preparative liquid chromatography (semi-pre HPLC).

[0012] The preparation method of the isoflavone compounds is carried out as follows:

[0013] <1> The stems of Derris trichocarpa are dried in the shade, crushed, sieved, added with ethanol solution, refluxed and extracted three times, each time for 1 hour, filtered and the filtrates are combined, the filtrates are concentrated under reduced pressure, and dried to obtain an ethanol extract;

[0014] <2> Step <1> The obtained extract is extracted with dichloromethane for multiple times, the extract is concentrated under reduced pressure, and dried to obtain a dichloromethane extract extract;

[0015] <3> Step <2> The obtained dichloromethane extract was separated by normal phase silica gel column chromatography, and gradient elution was performed with n-hexane-ethyl acetate system. All fractions were analyzed by high performance liquid chromatography, and fractions with the same components were combined and concentrated under reduced pressure.

[0016] <4> Step <3> The mid-gradient elution fraction is eluted by a reverse phase column using methanol / water with different volume fractions as the mobile phase, and then the reverse phase column elution fraction is prepared by liquid phase analysis.

[0017] step <1> The volume concentration of the ethanol solution is 80%, and the ethanol solution is added at a ratio of 5 times the volume weight of the ethanol solution to the stem of Derris trichocarpa.

[0018] step <3> The eluent for gradient elution was a mixture of n-hexane and ethyl acetate with a volume ratio of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:1.

[0019] step <4> The gradient elution fraction is the elution fraction of n-hexane-ethyl acetate mixture with a volume ratio of 3:1 (Fr.D1); the eluent for reverse phase column elution is methanol / water with a volume fraction of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%; the reverse phase column elution fraction is the first fraction (Fr.D1-4.1) in the elution fraction of methanol / water with a volume fraction of 40%; the conditions for liquid phase analysis preparation are detection wavelength of 260nm-290nm and volume fraction of 53% methanol / water.

[0020] The isoflavone compounds are used in the preparation of drugs for the treatment and prevention of hyperlipidemia and hypertriglyceridemia.

[0021] Hyperlipidemia and high triglycerides are insulin resistance type hyperlipidemia and high triglycerides.

[0022] In order to explore the medicinal value of Derris trichocarpa, the inventors conducted in-depth research and extracted and separated an isoflavone compound (hereinafter referred to as Compound 1) from the stem of Derris trichocarpa. Its chemical structure was determined by nuclear magnetic resonance hydrogen spectrum, carbon spectrum, high-resolution mass spectrometry and chemical methods as follows:

[0023]

[0024] The chemical name is: 6-(2-hydroxy-3-methyl-3-enyl)-3-(4-hydroxyphenyl)-5,7-dimethoxyisoflavone.

[0025] After comparison, it was determined that compound 1 was a new isoflavone compound, which was isolated from the traditional Chinese medicine Derris trichocarpa for the first time and has not been reported so far. Solubility experiments showed that the compound was easily soluble in organic solvents such as ethanol. At the same time, the inventors also established a corresponding preparation method, extracting the stems of Derris trichocarpa with ethanol solution, and then analyzing the extract by high-performance liquid chromatography, and then using a n-hexane-ethyl acetate elution system for gradient elution, and further separating by reverse phase column chromatography and semi-preparative liquid chromatography.

[0026] The lipid-lowering activity experiment proved that compared with the model group, the positive drug group and the high, medium and low dose groups of the compound 1 of the present invention can significantly reduce the TG content (P < 0.01), and each dose group of compound 1 shows a dose-dependency on insulin-resistant cells. The high and medium dose groups of compound 1 have a better effect on reducing the TG content than the positive drug group, and have the effect of reducing the TG content of insulin-resistant HepG2 cells. Therefore, the isoflavone compounds of the present invention have great development potential in the treatment and prevention of hyperlipidemia and high triglycerides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the isoflavone compound of the present invention (1 H-NMR).

[0028] Figure 2 is the carbon NMR spectrum of the isoflavone compounds of the present invention ( 13 C-NMR).

[0029] Figure 3 It is the high resolution mass spectrometry (HRMS) of the isoflavone compounds of the present invention. DETAILED DESCRIPTION

[0030] 1. Preparation of Compound 1

[0031] (1) The stems of Derris trichocarpa are dried in the shade, crushed, sieved, and 5 volumes of 80% ethanol are added to extract Derris trichocarpa powder under reflux. The extraction is performed three times, each time for 1 hour, filtered, and the filtrates are combined. The above operation is repeated three times. A total of 5 kg of Derris trichocarpa powder is obtained. The extraction filtrate is concentrated under reduced pressure and dried to obtain 0.4 kg of crude extract (ethanol extract);

[0032] (2) repeatedly extracting the extract obtained in step (1) with dichloromethane, concentrating the extract under reduced pressure, and drying to obtain 33 g of dichloromethane extract of Derris tricholoma;

[0033] (3) The dichloromethane extract of Derris tricholoma obtained in step (2) was separated by normal phase silica gel column chromatography, and gradient eluted with n-hexane-ethyl acetate in different ratios (V / V) of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, and 0:1 to obtain 21 fractions, each of which was concentrated under reduced pressure, and analyzed for components by high performance liquid chromatography, and then the fractions with the same components were combined and further separated and purified.

[0034] (4) The n-hexane-ethyl acetate (3:1) elution fraction (Fr.D1) was eluted by reverse phase column chromatography with methanol / water eluent of different volume fractions (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%). The preparation conditions of the first fraction (Fr.D1-4.1) in methanol / water (40%) were analyzed by liquid phase analysis, and the detection wavelength was determined to be 285 nm. 53% methanol / water was used as the preparation conditions for the preparation of the liquid phase to obtain compound 1.

[0035] 2. Structural confirmation of compound 1

[0036] 1. Structure Determination

[0037] The compound is white flaky crystals.

[0038] H NMR 1 H NMR (800 MHz, Chloroform-d) data ( Figure 1): δ7.80 (d, J=2.3Hz, 1H), 7.34 (d, J=2.0Hz, 1H), 7.33 (d, J=2.7Hz, 2H), 6.86 (d, J= 2.0Hz, 1H), 6.85 (d, J=2.5Hz, 2H), 6.67 (d, J=2.6Hz, 1H), 4.92 (s, 1H), 4.81-4.79 (m , 1H), 4.25 (d, J=4.0Hz, 1H), 4.24 (d, J=4.2Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.03 (d, J=4.0Hz, 1H), 3.01 (d, J=4.1Hz, 1H), 2.90 (dd, J=13.6, 8.9Hz, 1H), 1.83 (s, 3H).

[0039] C NMR 13 C NMR (201 MHz, Chloroform-d) data ( Figure 2 ): δ175.41, 162.38, 150.73, 147.50, 130.54, 126.21, 123.30, 119.43, 115.53, 112.98, 110.44, 95.61, 62.34, 56.19, 29.84, 18.07.

[0040] High-resolution mass spectrometry of compounds ( Figure 3 ): HR-ESI-MS(m / z 383.1482[M+H] + ).

[0041] 2. Structural derivation

[0042] (1) Molecular formula: C 22 H 22 O6.

[0043] (2) Chemical structure:

[0044]

[0045] III. Determination of the lipid-lowering and triglyceride-lowering activity of compound 1

[0046] 1. Experimental methods

[0047] 1.1 Cell culture

[0048] The HepG2 cells in good growth state were placed in a 37° C., 5% CO 2 incubator for culture. The culture medium used was a DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin double antibody.

[0049] 1.2 Establishment of hyperlipidemia cell model and in vitro lipid-lowering experiment

[0050] HepG2 cells in the logarithmic growth phase were evenly inoculated in a 6-well plate, 2 mL per well. After culturing in an incubator for 24 h, culture medium, oleic acid and drugs were added according to the experimental groups, and then cultured in an incubator for 12 h. Cell lysis buffer was added and lysed on ice for 10 min. The intracellular TG (triglyceride) content was determined according to the kit instructions.

[0051] 2. Experimental results

[0052] Table 1 Effect of compound 1 on TG content in insulin-resistant HepG2 cells

[0053]

[0054] Note: Compared with the model group, * P<0.05, ** P<0.01; compared with the normal group, # P<0.05, ## P<0.01.

[0055] The experimental results of the effect of compound 1 on the TG content of insulin-resistant HepG2 cells showed that the TG content of the high-dose group and the medium-dose group of compound 1 decreased by 43.91% and 23.74%, respectively, and the TG content of the positive drug group decreased by 38.38%. The effect of compound 1 in reducing TG content was better than that of the positive drug group, indicating that compound 1 has a good effect in reducing the TG content of insulin-resistant HepG2 cells.

[0056] Based on the above analysis, compound 1 is a new pure natural plant active ingredient with good TG-lowering effect, and can be further developed as a new drug for lowering insulin resistance-type hyperlipidemia and hypertriglyceridemia.

Claims

1. An isoflavone compound, characterized in that The chemical structure is:

2. The method for preparing isoflavone compounds according to claim 1, characterized in that Proceed as follows: <1> The stems of Derris trichocarpa are dried in the shade, crushed, sieved, added with ethanol solution, refluxed and extracted three times, each time for 1 hour, filtered and the filtrates are combined, the filtrates are concentrated under reduced pressure, and dried to obtain an ethanol extract; <2> Step <1> The obtained extract is extracted with dichloromethane for multiple times, the extract is concentrated under reduced pressure, and dried to obtain a dichloromethane extract extract; <3> Step <2> The obtained dichloromethane extract is separated by normal phase silica gel column chromatography, and gradient elution is performed with a n-hexane-ethyl acetate system, all fractions are analyzed by high performance liquid chromatography, and fractions with the same components are combined, and the fractions are concentrated under reduced pressure; the eluent for the gradient elution is a n-hexane-ethyl acetate mixture with a volume ratio of 20:1, 10:1, 5:1, 3:1, 1:1, 1:3, 0:1; <4> Step <3> The medium gradient elution fraction is eluted by a reverse phase column using methanol / water with different volume fractions as the mobile phase, and then the reverse phase column elution fraction is prepared by liquid phase analysis; the gradient elution fraction is an elution fraction of a mixture of n-hexane and ethyl acetate with a volume ratio of 3:1; the eluent for the reverse phase column elution is methanol / water with a volume fraction of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%; The reverse phase column elution fraction is the first fraction in the elution fraction of methanol / water with a volume fraction of 40%; the conditions for the liquid phase analysis preparation are a detection wavelength of 260nm-290nm and a volume fraction of 53% methanol / water.

3. The method for preparing isoflavone compounds according to claim 2, characterized in that step <1> The volume concentration of the ethanol solution is 80%, and the ethanol solution is added at a ratio of 5 times the volume weight of the ethanol solution to the stem of Derris trichocarpa.

4. Use of the isoflavone compounds according to claim 1 in the preparation of drugs for the treatment and prevention of hyperlipidemia and hypertriglyceridemia; the hyperlipidemia and hypertriglyceridemia are insulin resistance type hyperlipidemia and hypertriglyceridemia.

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