A 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound, a preparation method and application thereof

Abstract

The present application relates to the technical field of natural medicine, and more particularly to 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 of the present application has a structure shown in formula I; ethanol and petroleum ether are used to extract hypericum first, and the petroleum ether extract is eluted by silica gel column chromatography, MCI resin column chromatography, Sephadex LH-20 gel column chromatography and ODS medium pressure column chromatography in sequence, and then the collected eluate is prepared by preparative ODS liquid chromatography and purified by semi-preparative ODS liquid chromatography to obtain the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound.The present application realizes the extraction of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound from hypericum for the first time, and the compound has great development value as a new drug for preventing or treating calcified aortic valve disease.

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, anti-bacterial, 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] ​This invention provides a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound with the structural formula shown in Formula I:

[0007]

[0008] This invention also provides a method for preparing the above-mentioned 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound, comprising the following steps:

[0009] (1) Using the dried aerial parts of Hypericum as raw material, an ethanol extract was obtained by ethanol percolation and concentration. The ethanol extract was suspended in water to obtain a suspension. The suspension was extracted and concentrated by petroleum ether to obtain a petroleum ether extract.

[0010] (2) The petroleum ether extract was subjected to silica gel column chromatography gradient elution to obtain A to F; the eluent was a mixed solution of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate was 10:1 to 1:2.

[0011] (3) Select E and elute it with MCI resin column chromatography gradient to obtain E1 to E7; the eluent is a mixed solution of methanol and water, and the volume ratio of methanol to water is 30:70 to 90:10.

[0012] (4) Select E6 and elute it isocratically with a Sephadex LH-20 gel chromatography column to obtain E6-1 to E6-6; the eluent is methanol;

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

[0014] (6) Fr.5 was selected and prepared by ODS liquid chromatography to obtain crude extract; the mobile phase was acetonitrile and water, and the volume ratio of acetonitrile and water was 60:40.

[0015] (7) The crude extract was purified by semi-preparative ODS liquid chromatography to obtain a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound. The mobile phase was methanol-water, and the volume ratio of acetonitrile to water was 80:20.

[0016] Optionally, 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.

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

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

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

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

[0021] Optionally, during gradient elution in step (5), the volume ratio of methanol to water increases from 60:40 to 90:10 in a gradient, with an increase rate of 5 ratios per hour, a flow rate of 25 mL / min, and the eluent is collected every 1 hour, for a total elution time of 6 hours.

[0022] The present invention also provides the use of the above-mentioned 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compounds in the preparation of medicaments for the prevention or treatment of calcified aortic valve disease.

[0023] The present invention also provides a drug for the prevention or treatment of calcific aortic valve disease, the raw materials of which include the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound prepared in this invention and pharmaceutically acceptable excipients.

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

[0025] This invention is the first to achieve the extraction of a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound from St. John's wort, which has the ability to inhibit calcification of heart valves. This type of compound has great development value as a novel drug for the prevention or treatment of calcified aortic valve disease. The design concept of this compound also provides new ideas and approaches for the development of novel drugs for the prevention or treatment of calcified aortic valve disease. Attached Figure Description

[0026] Figure 1 The 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of this invention 1 H-NMR spectrum;

[0027] Figure 2 The 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of this invention 13 C1-NMR and DEPT spectra;

[0028] Figure 3 The graph shows the inhibition rate of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of this invention on the proliferation of valvular interstitial cells (VICs).

[0029] Figure 4The figure shows the effect of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of the present invention on the expression of ALP and RUNX2 in VICs cells cultured in osteogenic medium.

[0030] Figure 5 The effect of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of this invention on the alkaline phosphatase (ALP) activity in VICs cultured in osteogenic medium;

[0031] Figure 6 The effect of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of this invention on calcium salt deposition in VICs cells cultured in osteogenic medium;

[0032] Figure 7 The figure shows the effect of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of the present invention on the peak flow velocity and transvalvular pressure gradient of the aortic valve orifice in Apoe- / - mice. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0039] This invention provides a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound with the structural formula shown in Formula I:

[0040]

[0041] This invention also provides a method for preparing the above-mentioned 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound, comprising the following steps:

[0042] (1) Using the dried aerial parts of Hypericum as raw material, an ethanol extract was obtained by ethanol percolation and concentration. The ethanol extract was suspended in water to obtain a suspension. The suspension was extracted and concentrated by petroleum ether to obtain a petroleum ether extract.

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

[0044] (3) Select E and elute it with MCI resin column chromatography gradient to obtain E1 to E7; the eluent is a mixed solution of methanol and water, and the volume ratio of methanol to water is 30:70 to 90:10.

[0045] (4) Select E6 and elute it isocratically with a Sephadex LH-20 gel chromatography column to obtain E6-1 to E6-6; the eluent is methanol;

[0046] (5) Select E6-4 and elute it with an ODS medium-pressure column to obtain Fr.1 to Fr.6; the eluent is a mixed solution of methanol and water, and the volume ratio of methanol to water is 60:40 to 90:10.

[0047] (6) Fr.5 was selected and prepared by ODS liquid chromatography to obtain crude extract; the mobile phase was acetonitrile and water, and the volume ratio of acetonitrile and water was 60:40.

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

[0049] Step (1) of this invention uses the dried aerial parts of Hypericum as raw material, extracts it by ethanol percolation, concentrates the extract by rotary evaporation to obtain a paste-like ethanol extract, suspends the ethanol extract in water to obtain a suspension, extracts the suspension with petroleum ether, and concentrates the extract to obtain a paste, i.e., petroleum ether extract.

[0050] In this invention, 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 this invention, the volume ratio of petroleum ether to suspension is 1:1.

[0052] Step (2) of this invention involves gradient elution of the petroleum ether extract by silica gel column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate. During gradient elution, the eluent is eluted in the following order: volume ratio of petroleum ether to ethyl acetate is 10:1, 5:1, 3:1, 2:1, 1:1 and 1:2, to obtain A to F. The eluent E obtained when the volume ratio of the mixed solution of petroleum ether and ethyl acetate is 1:1 is collected and proceeded to step (3).

[0053] In step (3) of this invention, E is eluted by gradient chromatography on an MCI resin column. The eluent is a mixture of methanol and water. 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 in sequence to obtain E1 to E7. The eluent E6 obtained when the methanol and water volume ratio is 80:20 is collected and proceeded to step (4).

[0054] In step (4) of this invention, E6 is selected and eluted isocratically using a Sephadex LH-20 gel chromatography column. The eluent for isocratizing is pure methanol, the elution rate is 1 mL / min, the elution time is 9 h, and the eluent is collected every 1.5 h to obtain E6-1 to E6-6. The eluent E6-4 obtained from the 4th elution time is collected and proceeded to step (5).

[0055] Step (5) of this invention involves selecting E6-4 and performing gradient elution on an ODS medium-pressure chromatographic column. The eluent for gradient elution is a mixture of methanol and water with a volume ratio of 60:40 to 90:10. During gradient elution, the volume ratio of methanol to water increases from 60:40 to 90:10 at a rate of 5 ratios per hour. The flow rate is 25 mL / min. The eluent is collected every 1 hour, and the total elution time is 6 hours, resulting in 6 eluents from Fr.1 to Fr.6. The eluent Fr.5 obtained at the 5th elution time is collected and proceeds to step (6).

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

[0057] Step (7) of this invention involves taking the crude extract and purifying it by semi-preparative ODS liquid chromatography to obtain a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound. The mobile phase is methanol-water, and the volume ratio of acetonitrile to water is 80:20.

[0058] The present invention also provides the use of the above-mentioned 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compounds in the preparation of medicaments for the prevention or treatment of calcified aortic valve disease.

[0059] The present invention also provides a medicament for the prevention or treatment of calcific aortic valve disease, the raw materials of which include the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound prepared in this invention and pharmaceutically acceptable excipients.

[0060] In this invention, the dosage form of the drug for preventing or treating calcified aortic valve disease is tablets, capsules, granules, oral liquids, powders, drops, or microcapsules.

[0061] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063]

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

[0065] (1) Using the dried aerial parts of St. John's wort (1 kg) as raw material, the raw material was percolated with 95% ethanol for 120 h at a flow rate of 0.5 L / h, and concentrated by rotary evaporation to obtain an ethanol extract in paste form. The ethanol extract was suspended in water to obtain a suspension, and petroleum ether of equal volume to the suspension was added for extraction. The extract was concentrated to obtain the petroleum ether extract.

[0066] (2) The petroleum ether extract was subjected to gradient elution by silica gel column chromatography, with the eluent being a mixture of petroleum ether and ethyl acetate; specifically, during gradient elution, the volume ratio of petroleum ether to ethyl acetate was 10:1, 5:1, 3:1, 2:1, 1:1 and 1:2 in sequence, and 6 eluents (A to F) were collected in fractions after gradient elution with the eluent.

[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(logε): 201 (3.53) nm, 270 (2.68) nm; IR (potassium bromide) ν max :3430,2962,2922,2874,2852,1769,1746,1699,1459,1381,1142,1114cm –1 ;(+)-HRESIMS m / z 497.2508[M+Na] + (C 27 H 38 O7Na + The calculated value is m / z 497.2515; 1 H and 13 CNMR data can be found Figure 1 , Figure 2 See Table 1.

[0076] Table 1. Compounds of Formula I 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150MHz, CDCl3) data

[0077]

[0078] Example 3 Pharmacological Activity

[0079] Experimental methods and results

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

[0081] Human valvular interstitial cells (hVICs) were isolated from non-calcified aortic valve tissue. After three washes with PBS, the cells were digested with 1 mg / mL type I collagenase 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% CO2 humidification). Cells from passages 3-5 were used for subsequent experiments.

[0082] (2) Effect of Formula I compound on the proliferation of human valvular interstitial cells (VICs) by CCK-8 assay

[0083] The CCK8 kit was purchased from Absin (abs50003). Human cardiac valvular interstitial cells were injected at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of (cells / well) in 96-well plates. After serum starvation for 24 h, cells were treated with different concentrations (8–800 μM) of compound I for 72 h, followed by incubation in CCK-8 working solution (37 °C for 2 h). The absorbance at 450 nm was measured, and the IC50 of compound I was calculated. 50Value. The formula for calculating the inhibition rate of human cardiac valve interstitial cell growth by the test compound is: Cell growth inhibition rate % = (A (negative control group) - A (administered group)) / A (negative control group) × 100%

[0084] Experimental data analysis was performed using GraphPad Prism 8 statistical software, and the cell proliferation inhibitory activity of the samples was assessed using the half-maximal inhibitory concentration (IC50).

[0085]

[0086] AG represents the average OD value of the drug-treated group, AK represents the average OD value of the blank control group, and AM represents the average OD value of the model group.

[0087] Compared with the control group, compound I inhibited the growth of valvular interstitial cells in a dose-dependent manner, IC50 50 The value was 147.7 μmol / L. Figure 3 ).

[0088] (3) Compound I can inhibit osteogenic differentiation of valve interstitial cells.

[0089] Valvular interstitial cells were collected, homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (cocktails) on ice, and centrifuged at 12,000 rpm (4°C, 15 min) to collect the supernatant. Equal volumes of protein were subjected to SDS-PAGE electrophoresis, with the target protein being electrophoresed in 4–20% Tris-Glycine Mini Gels, and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked for 1 hour at room temperature with TBS-T buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween-20) containing 5% skim milk powder, followed by incubation overnight at 4°C with primary antibody. The membrane was then washed with TBS-T and incubated for 1 hour at room temperature with secondary antibody labeled with the corresponding Formula I compound. The immune complexes were then visualized using enhanced chemiluminescence (ECL) reagent. GAPDH was used as a standard for total protein assay. Specific bands were quantified by densitometry using Image J 1.54.

[0090] In the alkaline phosphatase (ALP) staining experiment, valvular interstitial cells were co-incubated with the above intervention in conditioned medium for 7 days. The cultured valvular interstitial cells were washed twice with PBS and fixed with 4% paraformaldehyde (PEA) for 10 minutes at room temperature. ALP staining was then performed using the BCIP / NBT alkaline phosphatase staining kit (Beyotime), following the kit's instructions. In short, ALP staining solution was added, incubated at room temperature for 20 minutes in the dark, and then the staining solution was removed, followed by rinsing the cell monolayer 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] Based on dose-response curves and IC50 50 In subsequent experiments, cells were treated with compound I at a concentration of 40 μmol / L. In the alizarin red staining experiment, VICs were incubated for 21 days in different culture media. Calcium deposition was detected using alizarin red staining. The procedure was as follows: cells were washed twice with PBS, then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. Cells were washed three times with ddH2O, incubated with 0.2% alizarin red solution (Sigma-Aldrich) for 30 minutes, and then washed three times with ddH2O. Images were observed using an Olympus BX51 microscope and captured using an Olympus DP71 camera and CellSens software.

[0092] Male C57BL / 6J and apolipoprotein E gene knockout (Apoe- / -) mice (weighing 20-25g, 6 weeks old) were purchased from Wuhan Solarbio Biotechnology Co., Ltd. Wild-type control mice (C57BL / 6J, n=8) were fed a standard maintenance diet, while the Apoe- / - group (n=8) was fed a Western diet. After transthoracic echocardiography and hemodynamic assessment, the mice were euthanized, and aortic valves were collected for histopathological examination. In animal experiments to validate compound I, the Apoe- / - group was fed a Western diet for 6 weeks, during which time compound I (20mg / kg) was administered intraperitoneally every 72 hours.

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

[0094] In summary, the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound prepared in this invention exhibits excellent inhibitory activity against cardiac valve calcification and can be used to prepare drugs for calcified aortic valve disease. This type of compound has significant development value as a novel drug for the prevention or treatment of calcified aortic valve disease, and the design concept of this compound also provides new ideas and approaches for the development of novel drugs for the prevention or treatment of calcified aortic valve disease.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

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

2. The method for preparing the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound according to claim 1, characterized in that, Includes the following steps: (1) Using the dried aerial parts of Hypericum as raw material, an ethanol extract was obtained by ethanol percolation and concentration. The ethanol extract was suspended in water to obtain a suspension. The suspension was extracted and concentrated by petroleum ether to obtain a petroleum ether extract. (2) The petroleum ether extract was subjected to gradient elution by silica gel column chromatography to obtain A to F; the eluent was a mixed solution of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate was 10:1 to 1:

2. (3) Select E and elute it with MCI resin column chromatography gradient to obtain E1 to E7; the eluent is a mixed solution of methanol and water, and the volume ratio of methanol to water is 30:70 to 90:

10. (4) Select E6 and elute it isocratically with a Sephadex LH-20 gel chromatography column to obtain E6-1 to E6-6; the eluent is methanol; (5) Select E6-4 and elute it with an ODS medium-pressure column to obtain Fr.1 to Fr.6; the eluent is a mixed solution of methanol and water, and the volume ratio of methanol to water is 60:40 to 90:

10. (6) Fr.5 was selected and prepared by ODS liquid chromatography to obtain crude extract; the mobile phase was acetonitrile and water, and the volume ratio of acetonitrile and water was 60:40; (7) The crude extract was purified by semi-preparative ODS liquid chromatography to obtain a 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound. The mobile phase was methanol-water, and the volume ratio of acetonitrile to water was 80:

20.

3. The preparation method according to claim 2, characterized in that, 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.

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

1.

5. The preparation method according to claim 2, characterized in that, In step (2), during gradient elution, elution is performed in the following order: petroleum ether to ethyl acetate in volume ratios of 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), during gradient elution, elution is performed in the following order: methanol to water volume ratios of 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 during isocratic elution is 1 mL / min, the elution time is 9 h, and the eluent is collected every 1.5 h.

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

9. The use of the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound of claim 1 in the preparation of a medicament for the prevention or treatment of calcified aortic valve disease.

10. A drug for the prevention or treatment of calcific aortic valve disease, characterized in that, The raw materials include the 3 / 6 / 5 / 6 / 5 pentacyclic phloroglucinol compound as described in claim 1 and pharmaceutically acceptable excipients.

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