Hymoin A, a phloroglucinol heteroterpene compound, its preparation method and application

Abstract

The present application relates to a kind of resorcinol heteroterpene compound HymoinA and its preparation method and application, belong to the field of medicinal chemistry, the resorcinol heteroterpene compound HymoinA is first extracted from Hypericum monogynum Isolation, its structural formula is (I) formula.The compound shows excellent lipid-lowering activity in oleic acid-induced HepG2 cell, has the potential for application in the field of lipid-lowering drugs, the present application simultaneously relates to a kind of pharmaceutical composition, it includes above-mentioned resorcinol heteroterpene compound HymoinA, and pharmaceutically acceptable adjuvant, it has the effect of reducing lipid.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a phloroglucinol triterpenoid compound, Hymoin A, its preparation method, and its applications. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a type of hepatic steatosis caused by non-alcoholic factors and is a typical liver manifestation of metabolic syndrome. The incidence of fatty liver disease is showing an increasing trend year by year. According to relevant studies, the incidence of NAFLD in my country is about 15%, while in Europe and the United States it is over 20%. Studies have shown that the occurrence and development of NAFLD are closely related to total cholesterol (TC) and triglycerides (TG). Abnormal increases and accumulation of TC and TG can lead to lipid metabolism disorders and thus trigger NAFLD. Therefore, the discovery of drugs that inhibit TC and TG is of great research significance and value. Marketed NAFLD treatments, such as atorvastatin, fenofibrate, and metformin, have unsatisfactory treatment effects and significant side effects. Natural products, as one of the important sources of innovative drug discovery, have become a hot area in the treatment of NAFLD. Therefore, the discovery of new anti-NAFLD drugs from natural products has attracted the attention of many medicinal chemists. Phloroglucinol terpenes are natural products formed by the hybridization of a phloroglucinol core with multiple isopentenyl groups. These compounds often possess complex core structures such as rare bridged rings, spirorings, and adamantane. They have attracted considerable attention due to their complex and unique chemical structures and diverse biological activities. According to current literature, phloroglucinol terpenes exhibit significant physiological activities in anti-inflammatory, anti-tumor, antibacterial, anti-COVID-19, Alzheimer's disease, and heart failure treatments. However, research on their lipid-lowering effects is limited. Hypericum monogynum, a plant belonging to the genus Hypericum in the family Guttiferae, is rich in phloroglucinol terpenes and is widely distributed in southwestern China. Its flowers, roots, and leaves can all be used medicinally, possessing antirheumatic, hepatitis-treating, antibacterial, anti-inflammatory, bronchitis-treating, hemostatic, and tissue-regenerating effects. Currently, there is limited research on the chemical components of Hypericum perforatum, with the main components being phloroglucinols, spironolactones, flavonoids, and triterpenoids. Therefore, identifying novel phloroglucinol and triterpenoid compounds with lipid-lowering activity from the traditional Chinese medicine Hypericum perforatum is of significant research value and importance. Summary of the Invention

[0003] The purpose of this invention is to discover novel phloroglucinol-derived terpenoid compounds with lipid-lowering activity from the traditional Chinese medicine Hypericum perforatum, and to explore their application in the preparation of lipid-lowering drugs. This invention provides a phloroglucinol-derived terpenoid compound, its preparation method, and its application. The following technical solution is adopted to achieve the above objective:

[0004] Hymoin A, a phloroglucinol triterpenoid compound, has the structural formula (I):

[0005]

[0006] The method for preparing the phloroglucinol heteroterpene compound Hymoin A includes the following steps:

[0007] Step A: Take 9 kg of dried Hypericum flowers, use 50-70 kg of methanol as solvent, and extract 3-5 times at a temperature of 25-30℃, with each extraction lasting 3-5 days. Concentrate under reduced pressure to recover methanol and obtain crude extract A.

[0008] Step B: The crude extract A obtained in Step A was subjected to silica gel column chromatography, using a gradient elution of petroleum ether to ethyl acetate in a volume ratio of 100:0 to 0:100. Thin-layer chromatography was used to detect different fractions, and the fractions were combined according to their thin-layer chromatographic properties to obtain 7 fractions: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, and Fr.G.

[0009] Step C: Fraction Fr.B was separated using an RP-C18 column with a gradient elution of methanol to water at a volume ratio of 40:60 to 100:0. The fractions were combined according to thin-layer chromatography (TLC) results, yielding five subfractions: Fr.B1, Fr.B2, Fr.B3, Fr.B4, and Fr.B5. TLC colorimetry and liquid chromatography-mass spectrometry (LC-MS) confirmed that fraction Fr.B3 contained a phloroglucinol heteroterpene structure. Further analysis was performed using MC... Fr.B3 was separated using resin I with a gradient elution of methanol to water in a volume ratio of 60:40 to 100:0, yielding seven subfractions: Fr.B3-A, Fr.B3-B, Fr.B3-C, Fr.B3-D, Fr.B3-E, Fr.B3-F, and Fr.B3-G. Finally, Fr.B3-F was further purified by semi-preparative high-performance liquid chromatography to obtain the phloroglucinol heteroterpene compound Hymoin A.

[0010] The methanol used in the preparation method of the phloroglucinol triterpenoid compound Hymoin A is 100% industrial methanol.

[0011] In step A of the preparation method of the phloroglucinol triterpenoid compound Hymoin A, the crude extract is extracted by cold maceration.

[0012] In step B of the preparation method of the phloroglucinol triterpenoid compound Hymoin A, the concentrations of the petroleum ether / ethyl acetate gradient elution are successively 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100 (volume ratios).

[0013] In step C of the preparation method of the phloroglucinol heteroterpenoid compound Hymoin A, Fr. B is separated using an RP-C18 column with methanol / water gradient elution concentrations of 40:60, 50:50, 60:40, 70:30, 80:20, 85:15, 90:10, and 100:0 (volume ratios); Fr. B3 is separated using MCI resin with methanol / water gradient elution concentrations of 60:40, 70:30, 75:25, 80:20, 85:15, 90:10, and 100:0 (volume ratios).

[0014] In step C of the preparation method of the phloroglucinol triterpenoid compound Hymoin A, the packing material of the semi-preparative high-performance liquid chromatography column is reversed-phase octadecyl bonded silica gel.

[0015] The application of the phloroglucinol triterpenoid compound Hymoin A in the preparation of lipid-lowering drugs.

[0016] A pharmaceutical composition comprising a phloroglucinol triterpenoid compound, Hymoin A, and pharmaceutically acceptable excipients.

[0017] The pharmaceutical composition contains 0.1-99% by mass of phloroglucinol triterpenoid compound Hymoin A, with the remainder being a pharmaceutical carrier or excipient.

[0018] Experimental studies have shown that the phloroglucinol triterpenoid compound Hymoin A has a significant inhibitory activity on OA-induced TG accumulation in HepG2 cells, exhibiting a significant lipid-lowering effect. Moreover, its activity is stronger than that of the positive control drug atorvastatin, and it can be used to prepare lipid-lowering drugs. Attached Figure Description

[0019] Figure 1 This is the structural formula of the phloroglucinol heteroterpenoid compound Hymoin A of the present invention;

[0020] Figure 2 This is the 1H NMR spectrum of the phloroglucinol triterpenoid compound Hymoin A;

[0021] Figure 3 This is the carbon NMR spectrum of the phloroglucinol triterpenoid compound Hymoin A;

[0022] Figure 4It is the cytotoxicity of the phloroglucinol triterpenoid compound Hymoin A on oleic acid-induced HepG2 cells;

[0023] Figure 5 This describes the inhibitory effect of the phloroglucinol triterpenoid compound Hymoin A on oleic acid-induced TG accumulation in HepG2 cells. Detailed Implementation

[0024] Example 1

[0025] Hymoin A, a phloroglucinol triterpenoid compound, has the structural formula (I):

[0026]

[0027] The method for preparing the phloroglucinol heteroterpene compound Hymoin A includes the following steps:

[0028] Step A: Take 9 kg of dried Hypericum flowers, use 50-70 kg of methanol as solvent, and extract 3-5 times at a temperature of 25-30℃, with each extraction taking 3-5 days. Concentrate under reduced pressure to recover methanol and obtain 3 kg of crude extract A.

[0029] Step B: The crude extract A obtained in Step A was subjected to silica gel column chromatography, using a gradient elution of petroleum ether to ethyl acetate in a volume ratio of 100:0 to 0:100. Thin-layer chromatography (TLC) was used to detect different fractions, and the fractions were combined according to their TLC performance to obtain 7 fractions: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, and Fr.G.

[0030] Step C: Fraction Fr.B (80g) was separated using an RP-C18 column with a gradient elution of methanol to water at a volume ratio of 40:60 to 100:0. The fractions were combined according to TLC analysis to obtain five subfractions: Fr.B1, Fr.B2, Fr.B3, Fr.B4, and Fr.B5. Based on TLC colorimetric analysis and LC-MS detection, fraction Fr.B3 was confirmed to contain a phloroglucinol-based heteroterpene structure. Fr.B3 was separated using MCI resin with a gradient elution of methanol to water in a volume ratio of 60:40 to 100:0, yielding seven subfractions: Fr.B3-A, Fr.B3-B, Fr.B3-C, Fr.B3-D, Fr.B3-E, Fr.B3-F, and Fr.B3-G. Finally, Fr.B3-F was further purified by semi-preparative high-performance liquid chromatography (HPLC) to obtain Hymoin A (8 mg).

[0031] The methanol used in the preparation method of the phloroglucinol triterpenoid compound Hymoin A is 100% industrial methanol.

[0032] In step A of the preparation method of the phloroglucinol triterpenoid compound Hymoin A, the crude extract is extracted by cold maceration.

[0033] In step B of the preparation method of the phloroglucinol triterpenoid compound Hymoin A, the concentrations of the petroleum ether / ethyl acetate gradient elution are successively 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100 (volume ratios).

[0034] In step C of the preparation method of the phloroglucinol heteroterpenoid compound Hymoin A, Fr. B is separated using an RP-C18 column with methanol / water gradient elution concentrations of 40:60, 50:50, 60:40, 70:30, 80:20, 85:15, 90:10, and 100:0 (volume ratios); Fr. B3 is separated using MCI resin with methanol / water gradient elution concentrations of 60:40, 70:30, 75:25, 80:20, 85:15, 90:10, and 100:0 (volume ratios).

[0035] In step C of the preparation method of the phloroglucinol triterpenoid compound Hymoin A, the packing material of the semi-preparative high-performance liquid chromatography column is reversed-phase octadecyl bonded silica gel.

[0036] The application of the phloroglucinol triterpenoid compound Hymoin A in the preparation of lipid-lowering drugs.

[0037] A pharmaceutical composition comprising a phloroglucinol triterpenoid compound, Hymoin A, and pharmaceutically acceptable excipients.

[0038] The pharmaceutical composition contains 0.1-99% by mass of phloroglucinol triterpenoid compound Hymoin A, with the remainder being a pharmaceutical carrier or excipient.

[0039] Example 2

[0040] Structural identification of the phloroglucinol heteroterpenoid compound Hymoin A: This was achieved through high-resolution mass spectrometry (HRESIMS) and nuclear magnetic resonance spectroscopy. 1 HNMR, 13 Based on a comprehensive analysis of data including C NMR, 2D-NMR, infrared spectroscopy (IR), melting point (mp), and optical rotation, its physicochemical properties are as follows:

[0041] Hymoin A: Colorless crystals, mp 180-182℃, [α] 24 D +77.92 (c 0.15, MeOH); Molecular formula is C26 H 36 O5;IR(KBr)v max 1702,1615,1594,1376,1295,1114,1054,920cm -1 ;(+)-HRESIMS:m / z451.2446[M+Na] + (The calculated value is C) 26 H 36 O5Na, 451.2455). Nuclear magnetic resonance spectrum ( 1 H NMR, 13 (C NMR) is shown in the table below:

[0042]

[0043] Example 3

[0044] To further verify the beneficial effects of the compounds described in this invention, the inhibitory effect of HymoinA obtained in Example 1 on the accumulation of triglycerides (TG) in human liver cancer cells (HepG2) treated with oleic acid (OA) was used.

[0045] (1) Test sample:

[0046] Preparation of sample solutions: Accurately weigh an appropriate amount of Hymoin A and use DMSO to prepare sample solutions of different concentrations for pharmacological activity testing.

[0047] Cell line: The human liver-derived cell line HepG2 was obtained from the Chinese Academy of Sciences (Shanghai, China).

[0048] (2) Experimental principle: An OA-induced hyperlipidemic model of HepG2 cells was used as a screening model. When cells are stimulated by OA, lipids are deposited in the cells, resulting in a large accumulation of TG. Then, a TG content detection kit was used to measure the TG content of the control group and the experimental group to determine the inhibitory effect of the samples on TG accumulation.

[0049] (3) Test methods:

[0050] Cytotoxicity evaluation: HepG2 cells were seeded in 96-well cell culture plates (1 × 10⁶ cells per well) using the MTT assay. 4Cells were incubated at 37°C for 12 h, treated with OA (0.5 mM) for 24 h, and then different concentrations of Hymoin A (2, 4, 8 μM) were added to 96-well plates and cultured for 24 h. Then, 5 μL of MTT (5 mg / ml) solution was added to the remaining 100 μL of cell culture medium in the 96-well cell culture plate, and the plate was incubated in a 5% CO2 incubator for another 4 hours. Then, 150 μL of LDMSO was added and reacted for 10 min. The absorbance was measured at 490 nm using a microplate reader.

[0051] Evaluation of triglyceride (TG) accumulation: HepG2 cells in logarithmic growth phase were seeded into 6-well cell culture plates (3 × 10⁶ cells per well). 5 Cells were cultured (number of cells per cell), and after 12 hours, the cells in the culture plate were washed with PBS. A 0.5 mM oleic acid-bovine serum albumin (OA-BSA) complex was then added. Different concentrations of Hymoin A (2, 4, 8 μM) and a positive control (atorvastatin 2, 4, 8 μM) were then added to the culture medium, and the cells were cultured for 24 hours. Finally, a BSA-only culture medium was used as a control, and the accumulation of TG in the cells was measured using a TG assay kit to determine the inhibitory effect of the compounds on TG. The calculation formula is as follows:

[0052] Cell viability (%) = ([A 490nm ] OA+样品 / [A 490nm ] 空白 )×100

[0053] (4) Experimental Results: According to the above experiments, compound HymoinA did not exhibit cytotoxicity at three different concentrations (2, 4, and 8 μM). The results are shown in the table below. Figure 3 The inhibitory effect of Hymoin A on TG accumulation in OA-stimulated human hepatocellular carcinoma cells (HepG2) was measured (Note: Con., blank control group, DMSO (10 μM) only, no OA; Mod., model group, oleic acid (0.5 mM) added; Com.1, compound Hymoin A; ATO., positive control atorvastatin). The results are as follows: Figure 4 As shown (where Con., blank control group, only DMSO (10 μM) added, no OA added; Mod., model group, oleic acid (0.5 mM) added; Com.1, compound Hymoin A; ATO., positive control atorvastatin), at all three concentrations, Hymoin A significantly inhibited the accumulation of TG in OA-induced HepG2 cells, with a significantly stronger activity than the positive control drug, atorvastatin; in particular, the compound showed the strongest inhibitory effect at a concentration of 4 μM.

[0054] (5) Experimental conclusion: The compound HymoinA has a significant inhibitory activity on OA-induced TG accumulation in HepG2 cells, showing a significant lipid-lowering effect, and its activity is stronger than that of the positive control drug atorvastatin. Therefore, HymoinA of the present invention can be used to prepare lipid-lowering drugs.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that any other improvements and modifications made by researchers in this field without departing from the method and content of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A phloroglucinol-based heteroterpene compound, Hymoin A, has the structural formula (I): 。 2. A method for preparing the phloroglucinol heteroterpene compound Hymoin A as described in claim 1, characterized in that, Includes the following steps: Step A: Take 9 kg of dried Hypericum flowers, use 50-70 kg of methanol as solvent, and extract 3-5 times at a temperature of 25-30℃, with each extraction lasting 3-5 days. Concentrate under reduced pressure to recover methanol and obtain crude extract A. Step B: The crude extract A obtained in Step A was subjected to silica gel column chromatography, using a gradient elution of petroleum ether to ethyl acetate in a volume ratio of 100:0 to 0:

100. Thin-layer chromatography was used to detect different fractions, and the fractions were combined according to their thin-layer chromatographic properties to obtain 7 fractions: Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F, and Fr. G. Step C: Fraction Fr. B was separated using an RP-C18 column with a methanol-to-water gradient elution ratio of 40:60 to 100:

0. The fractions were combined according to thin-layer chromatography (TLC) results, yielding five subfractions: Fr. B1, Fr. B2, Fr. B3, Fr. B4, and Fr. B5. Based on TLC colorimetric analysis and liquid chromatography-mass spectrometry (LC-MS), fraction Fr. B3 was confirmed to contain phloroglucinol-based heteroterpenoid structures. Fr. B3 was then separated using MCI resin with a methanol-to-water gradient elution ratio of 60:40 to 100:0, yielding seven subfractions: Fr. B3-A, Fr. B3-B, Fr. B3-C, Fr. B3-D, Fr. B3-E, Fr. B3-F, and Fr. B3- Finally, G was further purified by semi-preparative high-performance liquid chromatography to obtain the phloroglucinol triterpenoid compound Hymoin A.

3. The method for preparing the phloroglucinol heteroterpene compound Hymoin A according to claim 2, characterized in that, In step A, the crude extract is extracted using cold maceration.

4. The method for preparing the phloroglucinol heteroterpene compound Hymoin A according to claim 2, characterized in that, In step B, the concentrations of the petroleum ether / ethyl acetate gradient elution are, in sequence, volume ratios of 100:0, 80:20, 70:30, 60:40, 50:50, and 0:

100.

5. The method for preparing the phloroglucinol heteroterpene compound Hymoin A according to claim 2, characterized in that, In step C, Fr. B was separated using an RP-C18 column, with methanol / water gradient elution concentrations of 40:60, 50:50, 60:40, 70:30, 80:20, 85:15, 90:10, and 100:0 (v / v). Fr. B3 was separated using MCI resin, with methanol / water gradient elution concentrations of 60:40, 70:30, 75:25, 80:20, 85:15, 90:10, and 100:0 (v / v).

6. The method for preparing the phloroglucinol heteroterpene compound Hymoin A according to claim 2, characterized in that, In step C, the column packing material for the semi-preparative high-performance liquid chromatography is reversed-phase octadecyl bonded silica.

7. The use of Hymoin A, a phloroglucinol triterpenoid compound as described in claim 1, in the preparation of lipid-lowering drugs.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the phloroglucinol triterpenoid compound Hymoin A as described in claim 1 and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition contains 0.1-99% by mass of the phloroglucinol triterpenoid compound Hymoin A as described in claim 1, with the remainder being a pharmaceutical carrier.

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