Hymoin B, a natural drug of the phloroglucinol triterpenoid class, its preparation method and application

Abstract

The present application relates to a kind of resorcinol heteroterpene compound Hymoin B and its preparation method and application, belong to the field of medicinal chemistry, the resorcinol heteroterpene compound Hymoin B is first extracted from Hypericum monogynum Isolation, its structural formula is (I) formula.The compound has obvious inhibitory effect on oleic acid (OA) 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 Hymoin B, and pharmaceutically acceptable adjuvant, it has the effect of lowering lipid.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry. Specifically, it relates to a phloroglucinol heteroterpenoid natural drug, Hymoin B, its preparation method, and its application. Specifically, it relates to Hymoin B, a heteroterpenoid natural drug with lipid-lowering activity extracted and isolated from Hypericum perforatum, its preparation method, and its application in lipid-lowering treatment. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a pathological syndrome characterized by diffuse macrovesicular steatosis of hepatocellular cells, caused by a variety of factors other than alcohol and other clearly defined liver-damaging factors. In recent years, with the accelerated pace of life, changing diets and lifestyles, and decreased physical activity, the incidence of NAFLD has been increasing. Some patients may develop cirrhosis or even liver failure, posing a significant threat to their health and lives. Related mechanistic studies have shown that large amounts of total cholesterol (TC) and triglycerides (TG) accumulate in the liver, leading to lipid metabolism disorders and inducing NAFLD. Meanwhile, existing NAFLD treatments, such as atorvastatin, fenofibrate, and metformin, have unsatisfactory therapeutic effects and significant side effects. Therefore, further research into highly effective, low-toxicity drugs that inhibit TC and TG is of significant research importance and value.

[0003] Phloroglucinol terpenes, as important components of natural products, have attracted considerable interest from medicinal chemists. They are particularly noteworthy due to their complex and unique chemical structures and diverse biological activities. They exhibit significant physiological activities in anti-inflammatory, anti-tumor, antibacterial, anti-Alzheimer's disease, and heart failure treatments. Therefore, the development and application of phloroglucinol terpenes in the pharmaceutical field hold broad prospects.

[0004] Hypericum monogynum, a semi-evergreen shrub belonging to the genus Hypericum in the family Guttiferae, is widely cultivated in the central and southern provinces of my country as both an ornamental plant and a traditional medicinal herb. According to the *Dictionary of Traditional Chinese Medicine*, its fruit can be used as a substitute for Forsythia suspensa, and its roots can be used to dispel wind-dampness, relieve coughs, and treat injuries. Due to its rich content of phloroglucinol-containing heteroterpenoids, numerous scholars have conducted extensive research on the chemical composition and pharmacological effects of Hypericum monogynum in recent years, yielding fruitful results. Therefore, further research into novel phloroglucinol-containing heteroterpenoids with lipid-lowering activity from Hypericum monogynum is of significant research value and importance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Hymoin B, a phloroglucinol triterpenoid compound extracted and isolated from St. John's wort for the first time, and its pharmaceutically acceptable salt in the preparation of lipid-lowering drugs.

[0006] To solve the technical problem of this invention, the following technical solution is provided:

[0007] Hymoin B, a phloroglucinol triterpenoid compound, has the structural formula (I):

[0008]

[0009] The method for preparing the phloroglucinol heteroterpene compound Hymoin B includes the following steps:

[0010] 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.

[0011] 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.

[0012] Step C: Fraction Fr.E was subjected to silica gel column chromatography with a gradient elution of petroleum ether to acetone at a volume ratio of 50:50 to 0:100. The fractions were combined according to the colorimetric characteristics of thin-layer chromatography to obtain 5 fractions: Fr.E1, Fr.E2, Fr.E3, Fr.E4, and Fr.E5. Fr.E3 was subjected to a second silica gel column chromatography with a gradient elution of petroleum ether to acetone at a volume ratio of 40:60 to 0:100. The fractions were combined to obtain 4 fractions: Fr.E3A, Fr.E3B, Fr.E3C, and Fr.E3D. Further, fraction Fr.E3C was subjected to semi-preparative high-performance liquid chromatography with acetonitrile and water at a volume ratio of 65:35 to obtain compound Hymoin B.

[0013] In step A, 100% methanol is used, and the extraction method is cold maceration extraction.

[0014] In step B, silica gel column chromatography, the gradient elution concentrations of petroleum ether / ethyl acetate are 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100 (volume ratios).

[0015] In step C, during the first silica gel column chromatography, the volume ratio of petroleum ether to acetone was 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively; during the second silica gel column chromatography, the volume ratio of petroleum ether to acetone was 40:60, 30:70, 20:80, 10:90, and 0:100, respectively.

[0016] In steps B and C, the colorimetric reagent used is anisaldehyde.

[0017] In step C, the column packing material for the preparative high-performance liquid HPLC is reversed-phase octadecyl bonded silica.

[0018] Application of Hymoin B, a phloroglucinol triterpenoid compound, in the preparation of lipid-lowering drugs.

[0019] The application of a pharmaceutically acceptable salt of a phloroglucinol triterpenoid compound, Hymoin B, in the preparation of lipid-lowering drugs.

[0020] A lipid-lowering drug composition prepared from compound Hymoin B or a pharmaceutically acceptable salt thereof and a carrier. Attached Figure Description

[0021] Figure 1 The structural formula of Hymoin B, a phloroglucinol-based heteroterpene compound of this invention, is shown below.

[0022] Figure 2 The 1H NMR spectrum of Hymoin B, a phloroglucinol triterpenoid compound of the present invention;

[0023] Figure 3 The carbon NMR spectrum of Hymoin B, a phloroglucinol triterpenoid compound of the present invention.

[0024] Figure 4 The study investigated the cytotoxicity of the phloroglucinol compound Hymoin B on oleic acid-induced HepG2 cells.

[0025] Figure 5 This study investigated the inhibitory effect of the phloroglucinol compound Hymoin B on oleic acid-induced TG accumulation in HepG2 cells. Detailed Implementation

[0026] Example 1

[0027] The chemical structural formula of Hymoin B referred to in the examples (the numbers in the formula are the positions of the carbon atoms in the chemical structure) is shown below:

[0028]

[0029] The preparation of the resorcinol heteroterpene compound Hymoin B specifically includes the following steps:

[0030] 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.

[0031] 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.

[0032] Step C: Fraction Fr.E was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether to acetone at a volume ratio of 50:50 to 0:100. The fractions were combined according to TLC colorimetric characteristics to obtain five fractions: Fr.E1, Fr.E2, Fr.E3, Fr.E4, and Fr.E5. Fr.E3 was then subjected to a second silica gel column chromatography, eluted with a gradient of petroleum ether to acetone at a volume ratio of 40:60 to 0:100, resulting in four fractions: Fr.E3A, Fr.E3B, Fr.E3C, and Fr.E3D. Finally, fraction Fr.E3C was eluted by semi-preparative high-performance liquid chromatography (HPLC) with acetonitrile and water at a ratio of 65:35 to obtain compound Hymoin B (9 mg).

[0033] In step A, the methanol used is 100% methanol;

[0034] In step A, the extraction method is cold maceration extraction;

[0035] In step B, during silica gel column chromatography, the gradient elution concentrations of petroleum ether / ethyl acetate were 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100 (volume ratios).

[0036] In step C, during the first silica gel column chromatography, the volume ratio of petroleum ether to acetone was 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively.

[0037] In this invention, during step C, the volume ratio of petroleum ether to acetone during the second silica gel column chromatography is 40:60, 30:70, 20:80, 10:90, and 0:100, respectively.

[0038] In steps B and C, the colorimetric reagent used is anisaldehyde colorimetric reagent;

[0039] In step C, the column packing material for the preparative high-performance liquid chromatography (HPLC) is reversed-phase octadecyl-bonded silica gel.

[0040] The application of a compound, phloroglucinol terpene, Hymoin B, in the preparation of lipid-lowering drugs.

[0041] The application of a pharmaceutically acceptable salt of a phloroglucinol triterpenoid compound, Hymoin B, in the preparation of lipid-lowering drugs.

[0042] The pharmaceutical composition contains the phloroglucinol triterpenoid compound Hymoin B as described in this invention and a pharmaceutically acceptable salt or an acceptable carrier or excipient;

[0043] To achieve the purpose of medication and enhance the therapeutic effect, the drug or drug composition of the present invention can be administered using any known method of administration.

[0044] Example 2

[0045] Structural identification of the phloroglucinol heteroterpenoid compound Hymoin B: High-resolution mass spectrometry (HRESIMS) and nuclear magnetic resonance spectroscopy were used for identification according to conventional methods. 1 H NMR, 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:

[0046] Hymoin B: Colorless oil, molecular formula C 26 H 38 O5;

[0047] Optical rotation: [α] 24 D +160.00 (c 0.10, MeOH);

[0048] Infrared spectrum IR: (KBr)v max :2921,1850,2359,1716,1688,1644,1469,1211,1184,1020,954,914,669cm -1 ;

[0049] High-resolution mass spectrometry (HR-ESI-MS): m / z 453.2608 [M+Na] + (calcd.for C 26 H 38 O5Na:453.2611);

[0050] Nuclear magnetic resonance spectroscopy (NMR) 1 H NMR, 13 (C NMR) is shown in the table below:

[0051]

[0052] Example 3

[0053] To further verify the beneficial effects of the present invention, the inhibitory effect of Hymoin B prepared in Example 1 on the accumulation of triglycerides (TG) in human liver cancer cells (HepG2) treated with oleic acid (OA) was used.

[0054] 1. Test sample:

[0055] Preparation of sample solutions: Accurately weigh an appropriate amount of Hymoin B and prepare sample solutions of different concentrations using DMSO for pharmacological activity testing.

[0056] Cell line: The human liver-derived cell line HepG2 was obtained from the Chinese Academy of Sciences (Shanghai, China).

[0057] 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, leading to a significant accumulation of triglycerides (TG). A TG content detection kit was then used to measure the TG content in the control and experimental groups to determine the inhibitory effect of the samples on TG accumulation.

[0058] 3. Test methods:

[0059] Cytotoxicity evaluation: HepG2 cells were seeded in 96-well cell culture plates (1 × 10⁶ cells per well) using the MTT assay. 4 Cells were incubated at 37°C for 12 h, treated with OA (0.5 mM) for 24 h, and then different concentrations of Hymoin B (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, 50 μL of LDMSO was added and reacted for 10 min. The absorbance was measured at 490 nm using a microplate reader.

[0060] 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 B (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:

[0061] Cell viability (%) = ([A 490nm ] OA+样品 / [A 490nm ] 空白 )×100

[0062] 4. Experimental Results: According to the above experiments, compound Hymoin B did not exhibit cytotoxicity at three different concentrations (2, 4, and 8 μM). The results are shown in the table below. Figure 4 As shown. We continued with subsequent experiments at this concentration to determine the inhibitory effect of Hymoin B on TG accumulation in OA-stimulated human hepatocellular carcinoma cells (HepG2). Results are shown below. Figure 5 As shown (Note: 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 B; ATO., positive control atorvastatin), when the concentration of compound Hymoin B was 2 and 4 μM, Hymoin B exhibited good lipid-lowering activity, significantly stronger than the positive control drug atorvastatin. In particular, when the concentration of compound Hymoin B was 4 μM, the TG content in cells was basically the same as that in the blank control.

[0063] 5. Experimental Conclusion: Compound Hymoin B exhibits significant inhibitory activity against OA-induced TG accumulation in HepG2 cells, demonstrating a marked lipid-lowering effect. At concentrations of 2 and 4 μM, its lipid-lowering activity is significantly stronger than that of the positive control drug atorvastatin. Therefore, the phloroglucinol terpene Hymoin B of this invention can be used to prepare lipid-lowering drugs.

[0064] 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 heteroterpene compound, Hymoin B, has the structural formula (I):

2. A method for preparing the phloroglucinol heteroterpene compound Hymoin B 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.E was subjected to silica gel column chromatography with a gradient elution of petroleum ether to acetone at a volume ratio of 50:50 to 0:

100. The fractions were combined according to the colorimetric characteristics of thin-layer chromatography to obtain 5 fractions: Fr.E1, Fr.E2, Fr.E3, Fr.E4, and Fr.E5. Fr.E3 was subjected to a second silica gel column chromatography with a gradient elution of petroleum ether to acetone at a volume ratio of 40:60 to 0:

100. The fractions were combined to obtain 4 fractions: Fr.E3A, Fr.E3B, Fr.E3C, and Fr.E3D. Further, fraction Fr.E3C was subjected to semi-preparative high-performance liquid chromatography with acetonitrile and water at a volume ratio of 65:35 to obtain compound Hymoin B.

3. The method for preparing the phloroglucinol heteroterpene compound Hymoin B according to claim 2, characterized in that, In step A, 100% methanol was used, and the extraction method was cold maceration extraction.

4. The method for preparing the phloroglucinol heteroterpene compound Hymoin B according to claim 2, characterized in that, In step B, during silica gel column chromatography, the gradient elution concentrations of petroleum ether / ethyl acetate were 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100, respectively (volume ratio).

5. The method for preparing the phloroglucinol heteroterpene compound Hymoin B according to claim 2, characterized in that, In step C, during the first silica gel column chromatography, the volume ratio of petroleum ether to acetone was 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively; during the second silica gel column chromatography, the volume ratio of petroleum ether to acetone was 40:60, 30:70, 20:80, 10:90, and 0:100, respectively.

6. The method for preparing the phloroglucinol heteroterpene compound Hymoin B according to claim 2, characterized in that, In steps B and C, the colorimetric reagent used is anisaldehyde.

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

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

9. The use of a pharmaceutically acceptable salt of the phloroglucinol triterpenoid compound Hymoin B as described in claim 1 in the preparation of a lipid-lowering drug.

10. A lipid-lowering drug composition, characterized in that, The lipid-lowering drug composition is prepared from compound Hymoin B or its pharmaceutically acceptable salt and carrier.

Images