A kind of isopentenyl phloroglucinol ether compound and its preparation method and application

Abstract

The present invention discloses an isopentenyl phloroglucinol ether compound and a preparation method and application thereof, and belongs to the technical field of natural medicine. Its structural formula is as follows: The present invention realizes the extraction of an isopentenyl phloroglucinol ether compound having the function of inhibiting heart valve calcification from Hypericum for the first time, and has a good inhibitory effect on heart valve calcification. This type of compound has great development value as a new type of calcific aortic valve disease drug, and the design idea of ​​this type of compound also provides a new idea and approach for the development of a new type of calcific aortic valve disease drug.

Description

Technical Field

[0001] The invention relates to the technical field of natural medicines, and in particular to an isopentenyl phloroglucinol ether compound and a preparation method and application thereof. Background Art

[0002] Calcific aortic valve disease (CAVD) is characterized by thickening and calcification of the aortic valve, which can develop into valvular sclerosis and rapidly progress to a series of abnormal hemodynamic changes, ultimately leading to heart failure and death. Currently, there is still a lack of effective drug treatment. Aortic valvular interstitial cells, as the main cell population of the aortic valve, are responsible for tissue homeostasis and valvular function, and play a key role in the occurrence and development of CAVD. Therefore, the proliferation and calcification of valvular interstitial cells in the early stage of CAVD are important targets for drug intervention and may effectively prevent the occurrence of heart valve calcification. Therefore, it is necessary to seek treatment methods and drugs with good efficacy and few toxic side effects. In recent years, traditional Chinese medicine has provided an important source and new opportunity for modern drug development. The active ingredients of natural products have become an important source of new drug research and development with their multi-target, high efficiency and low toxicity.

[0003] Hypericum wilfordii, the whole herb of the plant Hypericum wilfordii of the family Garcinia family. Many species of Hypericum wilfordii are medicinal plants. Traditional Chinese medicine believes that plants of this genus have the effects of clearing away heat and detoxifying, astringing and stopping bleeding, and removing dampness. It is used to treat hemoptysis, blood transformation, intestinal wind bleeding, traumatic bleeding, rheumatic bone pain, etc. In recent years, studies have found that plants of this genus have pharmacological activities such as anti-depression, anti-tumor, anti-viral, analgesic, antibacterial, and anti-inflammatory. Studies have shown that the chemical components of the traditional Chinese medicine Hypericum wilfordii mainly include two categories: phloroglucinol and ketone. Isopentenyl phloroglucinol is an important chemical component of the traditional Chinese medicine Hypericum wilfordii, and is also considered to be the main material basis for the many pharmacological activities of Hypericum wilfordii. However, there have been no patents or literature reports on isopentenyl phloroglucinol ether compounds and their heart valve calcification inhibitory activity so far. Summary of the invention

[0004] The purpose of the present invention is to provide an isopentenyl phloroglucinol ether compound and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is an isopentenyl phloroglucinol ether compound, whose structural formula is as follows:

[0007]

[0008] The second technical solution of the present invention is a method for preparing the isopentenyl phloroglucinol ether compound, comprising the following steps:

[0009] (1) Using Hypericum perforatum as a raw material, extracting and concentrating to obtain an extract, suspending the extract in water to obtain a suspension, and then extracting and concentrating the extract with petroleum ether to obtain a petroleum ether extract;

[0010] (2) The petroleum ether extract is sequentially subjected to silica gel column chromatography gradient elution, MCI resin column chromatography gradient elution, Sephadex LH-20 gel column chromatography isocratic elution and ODS subcolumn chromatography gradient elution to obtain Fr.1 to Fr.6; Fr.2 is purified by ODS preparative liquid chromatography to obtain the isopentenyl pyrogallol ether compound.

[0011] The third technical solution of the present invention is the use of the isopentenyl phloroglucinol ether compound in the preparation of drugs for preventing and treating calcific aortic valve disease.

[0012] A fourth technical solution of the present invention is a drug for preventing and treating calcific aortic valve disease, comprising the isopentenyl phloroglucinol ether compound.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The present invention realizes the extraction of isopentenyl phloroglucinol ether compounds with the ability to inhibit heart valve calcification from Hypericum for the first time, and has a good inhibitory effect on heart valve calcification. Such compounds have great development value as new drugs for calcific aortic valve disease, and the design ideas of such compounds also provide new ideas and approaches for the development of new drugs for calcific aortic valve disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The isopentenyl phloroglucinol ether compound of the present invention 1 H-NMR spectrum.

[0017] Figure 2 The isopentenyl phloroglucinol ether compound of the present invention 13 C-NMR spectrum.

[0018] Figure 3 The graph is a graph showing the inhibition rate of the isopentenyl phloroglucinol ether compounds of the present invention on the proliferation of valvular interstitial cells (VICs).

[0019] Figure 4The immunoblot diagram (a) and the corresponding statistical diagram (b) show the effects of the isopentenyl phloroglucinol ether compounds of the present invention on the expression of ALP and Runx2 in VICs cultured in osteogenic medium.

[0020] Figure 5 The staining diagram (a) and the corresponding statistical diagram (b) show the effects of the isopentenyl phloroglucinol ether compounds of the present invention on the alkaline phosphatase (ALP) activity in VICs cultured in osteogenic medium. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0027] The embodiment of the present invention provides an isopentenyl phloroglucinol ether compound, the structural formula of which is as follows:

[0028]

[0029] The embodiment of the present invention also provides a method for preparing the isopentenyl phloroglucinol ether compound, comprising the following steps:

[0030] (1) Using Hypericum perforatum as a raw material, extracting and concentrating to obtain an extract, suspending the extract in water to obtain a suspension, and then extracting and concentrating the extract with petroleum ether to obtain a petroleum ether extract;

[0031] (2) The petroleum ether extract is sequentially subjected to silica gel column chromatography gradient elution, MCI resin column chromatography gradient elution, Sephadex LH-20 gel column chromatography isocratic elution and ODS subcolumn chromatography gradient elution to obtain Fr.1 to Fr.6; Fr.2 is purified by ODS preparative liquid chromatography to obtain the isopentenyl pyrogallol ether compound.

[0032] In some specific embodiments, the Hypericum is the dried aerial part of Hypericum perforatum;

[0033] The diafiltration extraction method is: diafiltration extraction with 95% ethanol for 120 hours at a flow rate of 0.5 L / h.

[0034] In some specific embodiments, the eluent for gradient elution of silica gel column chromatography is petroleum ether-ethyl acetate; the volume ratio of petroleum ether to ethyl acetate is 10:1 to 1:2. Preferably, the volume ratio of petroleum ether to ethyl acetate is 10:1, 5:1, 3:1, 2:1, 1:1, 1:2.

[0035] In some specific embodiments, the eluent for gradient elution of the MCI resin column chromatography is methanol-water; the volume ratio of the methanol to water is 30:70 to 90:10. Preferably, the eluate eluted with an eluent of petroleum ether and ethyl acetate in a volume ratio of 1:1 is used for gradient elution of the MCI resin column chromatography; the volume ratio of methanol to water is 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10.

[0036] In some specific embodiments, the eluent for isocratic elution of the Sephadex LH-20 gel column chromatography is methanol. Preferably, the eluate eluted with the eluent having a volume ratio of methanol to water of 80:20 is subjected to isocratic elution of the Sephadex LH-20 gel column chromatography.

[0037] In some specific embodiments, the conditions for isocratic elution of the Sephadex LH-20 gel column chromatography are: elution rate of 1 mL / min, elution time of 6 h, collection every 1 h, and 6 eluates collected by elution segmentation.

[0038] In some specific embodiments, the mobile phase of the ODS sub-column chromatography gradient elution is methanol-water; the volume ratio of methanol to water is 60:40 to 80:20. Preferably, the eluate of the sixth time period of Sephadex LH-20 gel column chromatography isocratic elution is used for ODS sub-column chromatography gradient elution; the volume ratio of methanol to water is 60:40 to 80:20, the gradient rises, the rising speed is 2 ratios per hour, the flow rate is 25mL / min, the total elution time is 10 hours, and 6 eluates Fr.1 to Fr.6 are collected segmentally by the eluent gradient elution.

[0039] In some specific embodiments, the conditions for purifying Fr.2 by ODS preparative liquid chromatography are: the mobile phase is acetonitrile-water; the volume ratio of acetonitrile to water is 40:60.

[0040] The embodiment of the present invention also provides the use of the isopentenyl phloroglucinol ether compound in the preparation of a drug for preventing and treating calcific aortic valve disease.

[0041] The embodiment of the present invention also provides a drug for preventing and treating calcific aortic valve disease, comprising the isopentenyl phloroglucinol ether compound. Preferably, it also comprises a pharmaceutically acceptable excipient. Preferably, the drug is in the form of tablets, capsules, granules, oral liquids, granules, pills or pellets.

[0042] Example 1

[0043] An isopentenyl phloroglucinol ether compound, the structural formula of which is as follows:

[0044]

[0045] The preparation method of the compound comprises the following steps:

[0046] 1 kg of the dried aerial parts of Hypericum perforatum was used as raw material, and the mixture was extracted by filtration with 95% ethanol for 120 h at a flow rate of 0.5 L / h. The extract was concentrated by rotary evaporation to obtain an ethanol extract in the form of an extract. The ethanol extract was suspended in water to obtain a suspension, and petroleum ether with an equal volume to the suspension was added for extraction. The extract solution was concentrated to obtain a petroleum ether extract.

[0047] The extract of the petroleum ether part is subjected to gradient elution through silica gel column chromatography to obtain the first eluate, the eluent is petroleum ether-ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 10:1 to 1:2. Specifically, the volume ratio of petroleum ether to ethyl acetate is 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and 6 eluates are collected segmentally through the gradient elution of the eluent.

[0048] The eluate eluted with an eluent of petroleum ether-ethyl acetate in a volume ratio of 1:1 is subjected to gradient elution on an MCI resin column chromatography to obtain a second eluate, wherein the eluent is methanol-water, and the volume ratio of the methanol to water is 30:70 to 90:10. Specifically, the volume ratio of the methanol to water is 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10, and 7 eluates are collected segmentally by gradient elution with the eluent.

[0049] The eluate eluted with the eluent having a volume ratio of methanol to water of 80:20 is subjected to isocratic elution through Sephadex LH-20 gel column chromatography to obtain a third eluate, wherein the eluent is pure methanol. Specifically, the elution rate of the eluent is 1 mL / min, the elution time is 6 h, and the eluate is collected every 1 h, and 6 eluates are collected segmentally after elution with the eluent.

[0050] The eluate eluted by the eluent in the sixth time period is subjected to gradient elution by ODS medium pressure column chromatography to obtain Fr.1 to Fr.6, and the mobile phase is methanol-water, and the volume ratio of the methanol to water is 60:40 to 80:20. Specifically, the volume ratio of the methanol to water is 60:40 to 80:20 with a gradient increase, the increase rate is 2 ratios per hour, the flow rate is 25 mL / min, the total elution time is 10 hours, and the six eluates Fr.1 to Fr.6 are collected in sections by the eluent gradient elution.

[0051] Fr.2 was taken and subjected to ODS preparative liquid chromatography and eluted with a fixed ratio mobile phase to obtain an isopentenyl phloroglucinol ether compound, wherein the mobile phase was acetonitrile-water, and the volume ratio of acetonitrile to water was 40:60.

[0052] Example 2

[0053] The structure of the isopentenyl phloroglucinol ether compound prepared in Example 1 was identified.

[0054] The identification results are as follows:

[0055] The compound represented by formula I is a white amorphous powder, which is easily soluble in methanol and dichloromethane. A 10% sulfuric acid ethanol solution shows purple red. The compound represented by formula (I) was subjected to nuclear magnetic resonance and mass spectrometry (see Figure 1 , 2), optical rotation, infrared spectrum, ultraviolet spectrum and other data tests, thereby determining that the compound of formula I is the new isopentenyl phloroglucinol ether compound of the present invention.

[0056] Compound (Formula I): Colorless crystals; Melting point: 153–155°C; [α] 2 D 5 :–48.3 (methanol, c 0.1); UV (methanol) λ max (logε): 201(4.29)nm; IR (potassium bromide)ν max : 3408, 1968, 2921, 2851, 1762, 1734, 1466, 1382, 1104, 914cm –1 ;(+)-HRESIMS m / z 483.2352[M+Na] + (C 26 H 36 O7 + Calculated value for m / z 483.2359); 1 H and 13 C NMR data are shown in Table 1.

[0057] Table 1 Compounds of Formula I 1 H-NMR (600MHz, chloroform-d) and 13 C-NMR (150MHz, chloroform-d) data

[0058]

[0059]

[0060] The structural formula of the isopentenyl phloroglucinol ether compound prepared in Example 1 of the present invention is as follows:

[0061]

[0062] Example 3

[0063] Verification of the pharmacological activity of the isopentenyl phloroglucinol ether compounds prepared in Example 1 of the present invention:

[0064] 1. CCK-8 assay to detect the effect of the compound of formula I on the proliferation of valvular interstitial cells (VICs)

[0065] CCK8 kit was purchased from Bio-Tech. Aortic valve interstitial cells were cultured at 1×10 3The cells were seeded in a 96-well plate at a density of 1.5 cells / well. After 24 hours, the cells were treated with different concentrations (30, 125, 250, 500, 2000 μM) of the isopentenyl phloroglucinol ether compounds prepared in Example 1 for 72 hours, stained according to the instructions of the kit, and the absorbance at 450 nm was measured.

[0066] The experimental data were analyzed using GraphPad Prism 8 statistical software, and the cell proliferation inhibition activity of the samples was evaluated using the half-maximal inhibitory concentration. The calculation formula for the valvular interstitial cell growth inhibition rate of the test compound is:

[0067]

[0068] Among them, A G is the average OD value of the drug-treated group, A K is the average OD value of the blank control group, A M is the average OD value of the model group.

[0069] The results are as follows Figure 3 As shown, compared with the control group, the isopentenyl phloroglucinol ether compound prepared in Example 1 inhibited the growth of valvular interstitial cells in a dose-dependent manner, IC 50 The value was 207.3 μM, and there was no obvious cytotoxicity to valvular interstitial cells within 100 μmol / L.

[0070] 2. Formula I compounds can inhibit the osteogenic differentiation of valvular interstitial cells

[0071] Valvular interstitial cells were collected and homogenized with RIPA lysis buffer containing protease and phosphatase inhibitor cocktails. Equal amounts of protein were subjected to SDS-PAGE electrophoresis in 4-20% Tris-Glycine Mini Gels for 40-60 min depending on the target protein and then transferred to polyvinylidene difluoride membranes. Blocking was performed with TBS-T (50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% Tween-20) buffer containing 5% skim milk powder for 1 hour at room temperature and then incubated with primary antibodies at 4°C overnight. Subsequently, the cells were washed with TBS-T and incubated with HRP-labeled secondary antibodies for 1 hour at room temperature. The immune complexes were then visualized using enhanced chemiluminescence (ECL) reagents. GAPDH was used as a standard for total protein determination. Specific bands were quantified by optical density determination using image J 1.54. For alkaline phosphatase (ALP) staining experiments, valvular interstitial cells were incubated with the above interventions in conditioned medium for 7 days. ALP staining was used to detect ALP expression. The cultured valvular interstitial cells were washed twice with PBS and fixed with 4% PEA for 15 minutes at room temperature. ALP staining was then performed using an alkaline phosphatase staining kit, and the operation steps were performed according to the kit instructions. ALP staining solution was added and incubated at room temperature for 20 minutes, protected from light, and then the staining solution was removed and the cell monolayer was rinsed 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.

[0072] After 3 days of treatment with the compound of formula I, Western blotting revealed that the compound of formula I could significantly reduce the expression levels of osteoblast markers ALP and Runx2 (see 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 valvular interstitial cells (see Figure 5 ).

[0073] In summary, the present invention has achieved the extraction of isopentenyl phloroglucinol ether compounds that inhibit the calcification of cardiac valve cells from Hypericum for the first time. Such compounds have great development value as new drugs for calcific aortic valve disease, and the design ideas of such compounds also provide new ideas and approaches for the development of new drugs for calcific aortic valve disease.

[0074] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An isopentenyl phloroglucinol ether compound, characterized in that: Its structural formula is as follows:

2. The method for preparing the isopentenyl phloroglucinol ether compound according to claim 1, characterized in that: The following steps are involved: (1) Using Hypericum perforatum as a raw material, extracting and concentrating to obtain an extract, suspending the extract in water to obtain a suspension, and then extracting and concentrating the extract with petroleum ether to obtain a petroleum ether extract; (2) The petroleum ether extract is sequentially subjected to silica gel column chromatography gradient elution, MCI resin column chromatography gradient elution, Sephadex LH-20 gel column chromatography isocratic elution and ODS medium pressure column chromatography gradient elution to obtain Fr.1 to Fr.6; Fr.2 is purified by ODS preparative liquid chromatography to obtain the isopentenyl pyrogallol ether compound according to claim 1.

3. The preparation method according to claim 2, characterized in that: The Hypericum is the dried aerial part of Hypericum; The diafiltration extraction method is: diafiltration extraction with 95% ethanol for 120 hours at a flow rate of 0.5 L / h.

4. The preparation method according to claim 2, characterized in that: The eluent for gradient elution of the silica gel column chromatography is petroleum ether-ethyl acetate; the volume ratio of the petroleum ether to ethyl acetate is 10:1 to 1:

2.

5. The preparation method according to claim 2, characterized in that: The eluent for gradient elution of the MCI resin column chromatography is methanol-water; the volume ratio of the methanol to water is 30:70-90:

10.

6. The preparation method according to claim 2, characterized in that: The eluent for isocratic elution of the Sephadex LH-20 gel column chromatography is methanol.

7. The preparation method according to claim 2, characterized in that: The mobile phase of the ODS medium-pressure column chromatography gradient elution is methanol-water; the volume ratio of the methanol to water is 60:40-80:

20.

8. The preparation method according to claim 2, characterized in that: The conditions for purifying Fr.2 by ODS preparative liquid chromatography are as follows: the mobile phase is acetonitrile-water; the volume ratio of acetonitrile to water is 40:

60.

9. Use of the isopentenyl phloroglucinol ether compound according to claim 1 in the preparation of a medicament for preventing and treating calcific aortic valve disease.

10. A drug for preventing and treating calcific aortic valve disease, characterized in that: It includes the isopentenyl phloroglucinol ether compound as described in claim 1.

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