3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of natural medicines, in particular to a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound as well as a preparation method and application thereof. The 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound disclosed by the invention has a structure as shown in a formula I; the method comprises the following steps: extracting hypericum monogynum by using ethanol and petroleum ether, sequentially eluting the obtained petroleum ether extract through silica gel column chromatography, MCI resin column chromatography, Sephadex LH-20 gel column chromatography and ODS medium-pressure column chromatography, and purifying the collected eluate through preparative ODS liquid chromatography and semi-preparative ODS liquid chromatography to obtain the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound. The 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound is extracted from hypericum monogynum for the first time, and the compound has great development value as a novel medicine for preventing or treating calcified aortic valve diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural medicine, and particularly relates to a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound and a preparation method and application thereof. BACKGROUND

[0002] Calcific aortic valve disease (CAVD) is a degenerative disease characterized by lipoprotein deposition, chronic inflammatory response and progressive fibrocalcification of the valve. The pathological core is the abnormal osteogenic differentiation of valve interstitial cells and extracellular matrix remodeling. Epidemiological surveys show that the incidence of this disease in people over 65 years old is 6%, and in people over 75 years old, it increases to 12%, which has become an important inducement of cardiovascular events in the elderly. Current clinical treatment mainly uses invasive treatment such as SAVR / TAVR, but there is a lack of effective drug therapy to prevent early disease progression. Studies have found that active ingredients of traditional Chinese medicine have a significant anti-calcification effect through multi-target mechanism of action, including inhibition of the NF-κB inflammatory pathway, down-regulation of Runx2 osteogenic transcription factor expression, and regulation of calcium and phosphorus metabolism homeostasis, which opens up a new direction for the development of new anti-CAVD drugs. Compared with chemically synthesized drugs, traditional Chinese medicine compounds have the advantage of “multi-component-multi-target-multi-pathway” synergistic regulation, and have lower toxicity and side effects, which provides a unique path for the development of innovative drugs that can not only delay the progression of valve calcification but also protect valve function.

[0003] The traditional Chinese medicine Hypericum is the whole herb of Hypericum japonicum. Many species of Hypericum are medicinal plants. Traditional Chinese medicine believes that the plants of this genus have the effects of clearing heat and resolving toxicity, astringing and hemostasis, and dampness. They are used to treat hemoptysis, blood, enterovirus, trauma, and rheumatic pain. In recent years, research has found that the plants of this genus have anti-depression, anti-tumor, anti-virus, analgesic, antibacterial, and anti-inflammatory activities. Studies have shown that the chemical components of the traditional Chinese medicine Hypericum mainly include phloroglucinols and ketones. Polycyclic polyisoprenyl phloroglucinols are important chemical components of the traditional Chinese medicine Hypericum and are also considered to be the main material basis for the many pharmacological activities of Hypericum. So far, there has been no patent or literature reported on a new 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound and its heart valve calcification inhibition activity. SUMMARY

[0004] The present application aims to provide a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound and a preparation method and application thereof. The 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound has good heart valve calcification inhibition activity and can be used for preparing a calcific aortic valve disease drug.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] ​The application provides a 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound, and a structural formula is shown as formula I.

[0007]

[0008] The application further provides a preparation method of the 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound.

[0009] (1) taking the dried aboveground part of Hypericum asperum as raw material, performing ethanol infiltration extraction, and concentrating to obtain ethanol extract; and mixing the ethanol extract in water to obtain a suspension, and performing petroleum ether extraction and concentration on the suspension to obtain a petroleum ether extract;

[0010] (2) performing gradient elution on the petroleum ether extract through a silica gel column to obtain A-F; an eluent is a mixed solution of petroleum ether and ethyl acetate, and a volume ratio of the petroleum ether to the ethyl acetate is 10:1 to 1:2;

[0011] (3) selecting E to perform gradient elution through an MCI resin column to obtain E1-E7; an eluent is a mixed solution of methanol and water, and a volume ratio of the methanol to the water is 30:70 to 90:10;

[0012] (4) selecting E6 to perform isocratic elution through a Sephadex LH-20 gel column to obtain E6-1-E6-6; an eluent is methanol;

[0013] (5) selecting E6-4 to perform gradient elution through an ODS medium-pressure column to obtain Fr.1-Fr.6; an eluent is a mixed solution of methanol and water, and a volume ratio of the methanol to the water is 60:40 to 90:10;

[0014] (6) selecting Fr.5 to perform preparation through a preparation ODS liquid chromatography to obtain a crude extract; a mobile phase is acetonitrile and water, and a volume ratio of the acetonitrile to the water is 60:40;

[0015] (7) performing semi-preparation ODS liquid chromatography preparation and purification on the crude extract to obtain the 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound, and a mobile phase is methanol-water, and a volume ratio of the acetonitrile to the water is 80:20.

[0016] Optionally, a volume fraction of the ethanol is 95%, and a time of the infiltration extraction is 120 h; and an infiltration extraction flow rate of the ethanol is 0.5 L / h.

[0017] Optionally, a volume ratio of the petroleum ether to the suspension is 1:1.

[0018] Optionally, in the gradient elution in step (2), elution is sequentially performed according to a volume ratio of the petroleum ether to the ethyl acetate of 10:1, 5:1, 3:1, 2:1, 1:1 and 1:2.

[0019] Optionally, in the gradient elution of step (3), the elution is performed in the order of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10 of the volume ratio of methanol to water.

[0020] Optionally, in the isocratic elution of step (4), the elution rate is 1 mL / min, and the elution time is 9 h, and the eluate is collected every 1.5 h.

[0021] Optionally, in the gradient elution of step (5), the volume ratio of methanol to water is increased from 60:40 to 90:10 at a rate of 5 ratios per hour, and the flow rate is 25 mL / min, and the eluate is collected every 1 h, and the total elution time is 6 h.

[0022] The application further provides application of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound in the preparation of a drug for preventing or treating calcified aortic valve disease.

[0023] The application further provides a drug for preventing or treating calcified aortic valve disease, which comprises the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound prepared by the application and a pharmaceutically acceptable excipient.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The application realizes extraction of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound with inhibition of heart valve calcification from Hypericum ascyron for the first time, and the compound has great development value as a new drug for preventing or treating calcified aortic valve disease, and the design idea of the compound provides a new idea and approach for development of a new drug for preventing or treating calcified aortic valve disease. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A curve of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of the application on inhibition of VICs (valvular interstitial cells) proliferation 1 H-NMR spectrum

[0027] Figure 2 C-NMR and DEPT spectrum of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of the application 13 C-NMR and DEPT spectrum of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of the application

[0028] Figure 3 A curve of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of the application on inhibition of VICs (valvular interstitial cells) proliferation

[0029] Figure 4Figure showing the effect of the 3 / 6 / 5 / 6 / 5 pentahydroxyphenol compound of the present invention on the expression of ALP and RUNX2 in VICs cells cultured in osteogenic medium;

[0030] Figure 5 Figure showing the effect of the 3 / 6 / 5 / 6 / 5 pentahydroxyphenol compound of the present invention on the activity of alkaline phosphatase (ALP) in VICs cultured in osteogenic medium;

[0031] Figure 6 Figure showing the effect of the 3 / 6 / 5 / 6 / 5 pentahydroxyphenol compound of the present invention on calcium salt deposition in VICs cells cultured in osteogenic medium;

[0032] Figure 7 Figure showing the effect of the 3 / 6 / 5 / 6 / 5 pentahydroxyphenol compound of the present invention on peak flow velocity and transvalvular pressure difference at the aortic valve orifice in Apoe- / - mice. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is not to be considered limiting of the application, as further aspects, features, and embodiments of the application will become apparent to those skilled in the art from the following detailed description, which is to be considered in connection with the accompanying drawings.

[0034] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0036] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples presented herein. The specification and examples given should be considered exemplary only, with the true scope and spirit of the application indicated by the following claims.

[0037] As used herein, the terms "comprise", "comprising", "including", "include", "contain", "containing", "have" and "having" or the like are open-ended, that is, meaning "including but not limited to".

[0038] The raw materials used in the present application can be obtained commercially or prepared by the prior art.

[0039] The present application provides a 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound, the structural formula is shown as formula I:

[0040]

[0041] The present application also provides a preparation method of the above-mentioned 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound, comprising the following steps:

[0042] (1) taking the dried aboveground part of Hypericum asperum as raw material, ethanol infiltration extraction, concentration to obtain ethanol extract, ethanol extract is suspended in water to obtain a suspension, the suspension is extracted with petroleum ether, concentrated to obtain petroleum ether extract;

[0043] (2) the petroleum ether extract is subjected to silica gel column chromatography gradient elution to obtain A~F; the eluent is a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether and ethyl acetate is 10:1~1:2;

[0044] (3) selecting E to be subjected to MCI resin column chromatography gradient elution to obtain E1~E7; the eluent is a mixed solution of methanol and water, and the volume ratio of the methanol and water is 30:70~90:10;

[0045] (4) selecting E6 to be subjected to Sephadex LH-20 gel chromatography column isocratic elution to obtain E6-1~E6-6; the eluent is methanol;

[0046] (5) selecting E6-4 to be subjected to ODS medium pressure chromatography column gradient elution to obtain Fr.1~Fr.6; the eluent is a mixed solution of methanol and water, and the volume ratio of the methanol and water is 60:40~90:10;

[0047] (6) selecting Fr.5 to be subjected to preparative ODS liquid chromatography to obtain crude extract; the mobile phase is acetonitrile and water, and the volume ratio of the acetonitrile and water is 60:40;

[0048] (7) the crude extract is subjected to semi-preparative ODS liquid chromatography to obtain 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound, and the mobile phase is methanol-water, and the volume ratio of the acetonitrile and water is 80:20.

[0049] The step (1) of the present application is to take the dried above-ground part of Hypericum asperum as raw material, to obtain the extract by ethanol percolation extraction, to obtain the ethanol extract by rotary evaporation concentration of the extract, to obtain the suspension by suspending the ethanol extract in water, to obtain the extract by petroleum ether extraction of the suspension, and to obtain the petroleum ether extract by concentration of the extract.

[0050] In the present application, the volume fraction of ethanol is 95%, the percolation extraction time is 120 h, and the percolation extraction flow rate of ethanol is 0.5 L / h.

[0051] In the present application, the volume ratio of petroleum ether to the suspension is 1:1.

[0052] The step (2) of the present application is to perform gradient elution of the petroleum ether extract by silica gel column chromatography, the eluent is a mixture of petroleum ether and ethyl acetate, during the gradient elution, the volume ratio of petroleum ether to ethyl acetate is sequentially 10:1, 5:1, 3:1, 2:1, 1:1 and 1:2, to obtain A-F, and the eluate obtained when the volume ratio of the mixed solution of petroleum ether and ethyl acetate is 1:1 is collected as E to enter step (3).

[0053] The step (3) of the present application is to perform gradient elution of E by MCI resin column chromatography, the eluent is a mixture of methanol and water, during the gradient elution, the volume ratio of methanol to water is sequentially 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10, to obtain E1-E7, and the eluate obtained when the volume ratio of methanol to water is 80:20 is collected as E6 to enter step (4).

[0054] The step (4) of the present application is to perform isocratic elution of E6 by Sephadex LH-20 gel column chromatography, the eluent is pure methanol during the isocratic elution, the elution speed is 1 mL / min during the elution, and the elution time is 9 h, the eluate is collected every 1.5 h, to obtain E6-1-E6-6, and the eluate obtained at the fourth time is collected as E6-4 to enter step (5).

[0055] The step (5) of the present application is to perform gradient elution of E6-4 by ODS medium-pressure column chromatography, the eluent of the gradient elution is a mixture of methanol and water with a volume ratio of 60:40-90:10, during the gradient elution, the volume ratio of methanol to water is gradiently increased from 60:40 to 90:10, the increasing speed is 5 ratios per hour, the flow rate is 25 mL / min, the eluate is collected every 1 h, the total elution time is 6 h, to obtain 6 eluates Fr.1-Fr.6, and the eluate obtained at the fifth time is collected as Fr.5 to enter step (6).

[0056] The step (6) of the present application is to prepare Fr.5 by preparing ODS liquid chromatography, to obtain a crude extract; the mobile phase is acetonitrile and water, and the volume ratio of the acetonitrile and water is 60:40.

[0057] The step (7) of the present application is to purify the crude extract by semi-preparative ODS liquid chromatography, to obtain 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compounds, and the mobile phase is methanol-water, and the volume ratio of the acetonitrile and water is 80:20.

[0058] The present application also provides the use of the above-mentioned 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compounds in the preparation of a drug for preventing or treating calcified aortic valve disease.

[0059] The present application also provides a drug for preventing or treating calcified aortic valve disease, and the raw material comprises the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compounds prepared by the present application and pharmaceutically acceptable adjuvants.

[0060] In the present application, the dosage form of the drug for preventing or treating calcified aortic valve disease is tablets, capsules, granules, oral liquids, granules, dripping pills or pellets.

[0061] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0062] Example 1

[0063]

[0064] The preparation method of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compounds of formula I is as follows:

[0065] (1) 95% ethanol is used to infiltrate and extract 1 kg of dried aboveground parts of Hypericum asperulum for 120 h at a flow rate of 0.5 L / h, and rotary evaporation is performed to obtain ethanol extract in the form of extract; the ethanol extract is suspended in water to obtain a suspension, and petroleum ether is added to the suspension in an amount equal to the volume of the suspension to extract the suspension; and the extracted solution is concentrated to obtain a petroleum ether extract;

[0066] (2) The petroleum ether extract is subjected to gradient elution by silica gel column chromatography, and the eluent is a mixture of petroleum ether and ethyl acetate; specifically, when gradient elution is performed, the petroleum ether and ethyl acetate are eluted in the order of volume ratios of 10:1, 5:1, 3:1, 2:1, 1:1 and 1:2, and 6 eluates (A-F) are collected by elution with the eluent gradient;

[0067] (3) The eluent (E) obtained by elution with petroleum ether-ethyl acetate volume ratio of 1:1 in step (2) is subjected to MCI resin column chromatography and gradient elution with a mixed solution of methanol and water. Specifically, during gradient elution, the methanol and water volume ratios are 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10 respectively. Seven eluents (E1 to E7) are collected in fractions after gradient elution with eluent.

[0068] (4) Take the eluent (E6) eluted with methanol-water volume ratio of 80:20 in step (3) and perform Sephadex LH-20 gel column chromatography. Elute with pure methanol isocratic. Specifically, the elution rate of the eluent is 1 mL / min, the elution time is 9 h, and the eluent is collected every 1.5 h. After elution with the eluent, 6 eluents (E6-1 to E6-6) are collected in segments.

[0069] (5) Take the eluent (E6-4) from the fourth time period in step (4) and perform ODS medium-pressure column chromatography. Elute with methanol-water gradient solvent. Specifically, the eluent is methanol and water in a volume ratio of 60:40 to 90:10, with a gradient increase of 5 ratios per hour and a flow rate of 25 mL / min. Collect once every 1 hour for a total elution time of 6 hours. Collect 6 eluents Fr.1 to Fr.6 in segments after gradient elution.

[0070] (6) The Fr.5 fraction in step (5) was prepared by preparative ODS liquid chromatography using acetonitrile-water solution with a volume ratio of 60:40 as the mobile phase, and then purified by semi-preparative ODS liquid chromatography using methanol-water solution with a volume ratio of 80:20 as the mobile phase to obtain compound I.

[0071] Example 2

[0072] The structure of the compound of formula I prepared in Example 1 was identified.

[0073] The results of the identification are as follows:

[0074] The compound shown in Formula I is a colorless oil (methanol) that is highly soluble in methanol and dichloromethane. Nuclear magnetic resonance (NMR), mass spectrometry (MS), optical rotation, infrared spectroscopy, ultraviolet spectroscopy, and circular dichroism (CDD) analyses were performed on the compound shown in Formula (I), confirming that the compound in Formula I is the novel 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of this invention.

[0075] Physicochemical data of compound I (HMA): colorless oily substance; +6.4 (methanol, c 0.1); UV (methanol)λ max(log ε): 201 (3.53) nm, 270 (2.68) nm; IR (KBr) v max : 3430, 2962, 2922, 2874, 2852, 1769, 1746, 1699, 1459, 1381, 1142, 1114 cm –1 ; (+)-HRESIMS m / z 497.2508 [M + Na] + (C 27 H 38 O7Na + Calcd for m / z 497.2515); 1 H and 13 CNMR data are listed in Table 1. Figure 1 , Figure 2 and Table 1.

[0076] Table 1.1H-NMR (600 MHz, CDC13) and13C-NMR (150 MHz, CDC13) data of compounds of formula I. 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13) data

[0077]

[0078] Example 3 Pharmacological activity

[0079] Test method and results

[0080] (1) Isolation and culture of human VICs

[0081] Human cardiac valve interstitial cells (hVICs) were isolated from non-calcified aortic valve tissue. After three washes with PBS, the cells were digested with 1 mg / mL collagenase type I at 37 °C for 12 hours. The cell suspension was centrifuged at 1000 rpm for 10 minutes, and the precipitated cells were seeded in high glucose DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) under standard culture conditions (37 °C, 5% CO2humidified environment). The 3rd-5th generation of cells were used for subsequent experiments.

[0082] (2) CCK-8 method for detecting the effect of compounds of formula I on the proliferation of human cardiac valve interstitial cells (VICs)

[0083] The CCK8 kit was purchased from Absin abs50003 company. Human cardiac valve interstitial cells were seeded in a 96-well plate at a density of 1 x 10 4 After serum starvation culture for 24 h, the cells were treated with different concentrations (8-800 μM) of compounds of formula I for 72 h, followed by the addition of CCK-8 working solution (incubated at 37 °C for 2 hours), and the absorbance value at 450 nm was measured to calculate the IC 50The human heart valve interstitial cell growth inhibition rate of the test compound was calculated according to the following formula: Cell growth inhibition rate % = (A (negative control group) - A (dosing group)) / A (negative control group) x 100%

[0084] The experimental data were analyzed using GraphPad Prism 8 statistical software, and the cell proliferation inhibition activity of the sample was evaluated using the half inhibitory concentration.

[0085]

[0086] AG is the average OD value of the dosing group, AK is the average OD value of the blank control group, and AM is the average OD value of the model group.

[0087] Compared with the control group, the compound of formula I inhibited the growth of valve interstitial cells in a dose-dependent manner, and the IC 50 The value is 147.7 μmol / L Figure 3 ).

[0088] (3) The compound of formula I can inhibit the osteogenic differentiation of valve interstitial cells

[0089] The valve interstitial cells were collected, homogenized in an ice bath with RIPA lysis buffer containing protease and phosphatase inhibitor cocktails, and the supernatant was collected after centrifugation at 12,000 rpm (4°C, 15 minutes). An equal amount of protein was subjected to SDS-PAGE electrophoresis, and the target protein was electrophoresed in 4-20% Tris-Glycine Mini Gels and then transferred to a polyvinylidene fluoride membrane (PVDF). After blocking with 5% skim milk-containing TBS-T (50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% Tween-20) buffer at room temperature for 1 hour, the primary antibody was incubated at 4°C overnight. Then, it was washed with TBS-T and incubated with the corresponding secondary antibody labeled with the compound of formula I at room temperature for 1 hour. Then, the immunocomplexes were visualized using enhanced chemiluminescence (ECL) reagents. GAPDH was used as a standard for total protein determination. The specific bands were quantified by densitometry using image J 1.54.

[0090] In the alkaline phosphatase (ALP) staining experiment, the valve interstitial cells were co-incubated with the above interventions in the conditioned medium for 7 days. The cultured valve interstitial cells were washed twice with PBS and fixed with 4% paraformaldehyde (PEA) at room temperature for 10 minutes. Then ALP staining was performed using the BCIP / NBT alkaline phosphatase staining kit (Bi Yun Tian) according to the kit instructions. Briefly, ALP staining solution was added and incubated at room temperature for 20 min in the dark, and then the staining solution was removed and the cell monolayer was washed with double distilled water. Image visualization was performed using an Olympus BX51 microscope, and image acquisition was performed using an Olympus DP71 camera and cellens software.

[0091] According to the dose-response curve and IC 50 value, in the following experiments, the cells were treated with the compound of formula I at a concentration of 40 μmol / L. In the alizarin red staining experiment, the VICs were incubated in different conditioned media for 21 d. Alizarin red staining was used to detect calcium deposition. The steps are as follows: washed twice with PBS, then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. Wash the cells with ddH2O 3 times, incubate with 0.2% alizarin red solution (Sigma-Aldrich) for 30 minutes, and then wash with ddH2O 3 times. Use an Olympus BX51 microscope to observe the images, and use an Olympus DP71 camera and CellSens software to take pictures.

[0092] Male C57BL / 6J and apolipoprotein E gene knockout (Apoe- / -) mice (body weight 20-25 g, 6 weeks old) were purchased from Wuhan Solabio Biotechnology Co., Ltd. The wild type control group (C57BL / 6J, n = 8) was fed with standard maintenance feed, and the Apoe- / - group (n = 8) was fed with western diet. After completing the transthoracic echocardiography and hemodynamic evaluation, the mice were euthanized and the aortic valve was collected for histopathological examination. In the animal experiment verification of compound I, the Apoe- / - group was fed with western diet for 6 weeks, and compound I (20 mg / kg) was injected intraperitoneally every 72 h during the period.

[0093] After 3 days of treatment with the compound of formula I, Western blotting found that the compound of formula I could significantly reduce the expression levels of osteoblast markers ALP and Runx2 Figure 4 ). In addition, alkaline phosphatase staining showed that after 7 days of culture in osteogenic medium, the compound of formula I significantly reduced the ALP activity of valve interstitial cells Figure 5 ). In the 21-day long-term OM intervention experiment, the alizarin red staining results showed that compound I could reduce the calcium salt deposition of human primary VICs cells Figure 6) In the Apoe- / - mouse high-fat feeding model, long-term intraperitoneal injection of compound I can reduce the peak flow rate and transvalvular pressure difference of the aortic valve of Apoe- / - mice Figure 7

[0094] In summary, the 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound prepared by the present application has good heart valve calcification inhibition activity, and can be used for preparing drugs for calcified aortic valve diseases. The compound as a new type of drug for preventing or treating calcified aortic valve diseases has great development value, and the design idea of the compound provides a new idea and way for the development of new drugs for preventing or treating calcified aortic valve diseases.

[0095] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound, characterized in that, The structural formula is shown as Formula I:

2. The process for the preparation of 3 / 6 / 5 / 6 / 5 pentacyclic emodin compound as claimed in claim 1, wherein, The method comprises the following steps: (1) taking the dried above-ground parts of Hypericum asperum as raw material, extracting by ethanol infiltration, and concentrating to obtain ethanol extract, and then mixing the ethanol extract in water to obtain a suspension, and then extracting the suspension by petroleum ether and concentrating to obtain petroleum ether extract; (2) subjecting the petroleum ether extract to gradient elution by silica gel column chromatography to obtain A~F; the eluent is a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 10:1~1:2; (3) selecting E for gradient elution by MCI resin column chromatography to obtain E1~E7; the eluent is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 30:70~90:10; (4) selecting E6 for isocratic elution by Sephadex LH-20 gel column chromatography to obtain E6-1~E6-6; the eluent is methanol; (5) selecting E6-4 for gradient elution by ODS medium-pressure column chromatography to obtain Fr.1~Fr.6; the eluent is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 60:40~90:10; (6) selecting Fr.5 for preparation by preparative ODS liquid chromatography to obtain crude extract; the mobile phase is acetonitrile and water, and the volume ratio of the acetonitrile to the water is 60:40; (7) subjecting the crude extract to semi-preparative ODS liquid chromatography for purification to obtain 3 / 6 / 5 / 6 / 5 pentacyclic depsidic compound, and the mobile phase is methanol-water, and the volume ratio of the acetonitrile to the water is 80:

20.

3. The method of claim 2, wherein, The volume fraction of the ethanol is 95%, the infiltration extraction time is 120 h, and the infiltration extraction flow rate of the ethanol is 0.5 L / h.

4. The preparation method according to claim 2, characterized in that, The volume ratio of the petroleum ether to the suspension is 1:

1.

5. The preparation method according to claim 2, characterized in that, In step (2), the gradient elution is performed in the order of the volume ratio of the petroleum ether to the ethyl acetate being 10:1, 5:1, 3:1, 2:1, 1:1 and 1:

2.

6. The preparation method according to claim 2, characterized in that, In step (3), the gradient elution is performed in the order of the volume ratio of the methanol to the water being 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:

10.

7. The preparation method according to claim 2, characterized in that, In step (4), the elution rate is 1 mL / min, and the elution time is 9 h, and the eluate is collected every 1.5 h.

8. The preparation method according to claim 2, characterized in that, In step (5), the volume ratio of the methanol to the water is increased from 60:40 to 90:10 at a rate of 5 ratios per hour, the flow rate is 25 mL / min, the eluate is collected every 1 h, and the total elution time is 6 h.

9. The 3 / 6 / 5 / 6 / 5 pentacyclic depsidic compound of claim 1 is used for preparing a drug for preventing or treating calcified aortic valve disease.

10. A medicament for preventing or treating calcific aortic valve disease, characterized by, The raw material comprises the 3 / 6 / 5 / 6 / 5 pentacyclic depsidic compound of claim 1 and pharmaceutically acceptable adjuvant.

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