Hypericumonates D-F, a kind of prenyl-α-pyrone dimer, and preparation method and application thereof
By extracting and isolating isopentenyl-α-pyranone dimers (Hypericumonates DF) from the branches and leaves of St. John's wort, the adverse reaction problem of existing anti-neuroinflammatory drugs has been solved, achieving a highly effective and safe anti-inflammatory effect.
Patent Information
- Application Number
- CN202510024459.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2026-01-16
- Estimated Expiration
- 2045-01-07
AI Technical Summary
Existing anti-neuroinflammatory drugs have adverse reactions or toxic side effects, and there is a lack of effective and safe natural drug solutions on the market.
Hypericumonates DF, an isopentenyl-α-pyranone dimer, was extracted and isolated from the branches and leaves of Hypericum. Compounds with anti-neuroinflammatory activity were prepared by multi-step chromatography and purification techniques for the preparation of anti-neuroinflammatory drugs.
Hypericumonates DF, an isopentenyl-α-pyranone dimer, significantly inhibited LPS-induced nitric oxide production in BV-2 cells, exhibiting a stronger anti-inflammatory effect than the positive control drug, and without cytotoxicity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of pharmaceutical chemistry. Specifically, it relates to Hypericumonates D-F, a natural medicine with anti-neuroinflammatory activity, extracted and separated from the branches and leaves of Hypericum japonicum, a preparation method thereof, and an application thereof in the treatment of neuroinflammation. BACKGROUND
[0002] Neuroinflammation is a condition observed in the central nervous system (CNS) under the stimulation of infection, toxic metabolites, trauma, or autoimmunity. During the stimulation process, neuroglial cells such as microglia and astrocytes are activated to release a large amount of inflammatory mediators, leading to abnormal reactions or damage of neural tissue. It has a certain protective effect, but excessive neuroinflammation can cause various neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. A series of anti-neuroinflammatory drugs on the market, such as Memantine and Tramiprosate, will produce various adverse reactions or toxic side effects in use. Therefore, it is very important to discover new anti-neuroinflammatory candidate drugs. Natural medicines play an irreplaceable important role in innovative drug discovery, and their unique novel structures and good biological activities will still play a key role in the future drug research and development field. Therefore, it has become an important way in this research field to find and discover anti-neuroinflammatory candidate drugs from natural medicines.
[0003] Isoprenyl-α-pyrone dimers are formed by the [2+2] cycloaddition reaction of the intramolecular double bond of two molecules of α-pyrone and dimethyl pyran ring. Due to its unique chemical structure and good biological activity, it has a broad prospect in the development and application in the field of medicine. The isoprenyl-α-pyrone dimers Hypericumonates D-F described in the present application are obtained by the [2+2] cycloaddition reaction of the intramolecular double bond of one molecule of pyran and the intramolecular double bond of another molecule of α-pyrone, and the two molecules are connected by a cyclobutane to form a 6 / 6 / 4 / 6 / 6 ring system nucleus. Such compounds have not been reported in Hypericum, and so far, only four pairs of isoprenyl-α-pyrone dimers have been reported from Hypericum plants (see references 1 and 2). Such compounds have good NO inhibitory activity on LPS-induced RAW264.7 cells [1,2] .
[0004] Hypericum monogynum is a semi-evergreen shrub belonging to the genus Hypericum in the family Guttiferae. It 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. Current research on the chemical components of Hypericum monogynum mainly focuses on compounds such as phloroglucinols, spironolactones, flavonoids, and triterpenoids. Therefore, identifying novel isopentenyl-α-pyranone dimers with anti-neuroinflammatory activity from Hypericum monogynum is of significant research value and importance.
[0005] References:
[0006] [1]B.Zhen,XYSuo,J.Dang,HLYue,YDTao,JJWang,L.Li,MBLin,Q.
[0007] Hou,WPWang,XLWang,JDJiang and TFJi,Hyperterpenoids A and B:
[0008] two pairs of unprecedented 6 / 6 / 4 / 6 / 6polycyclic cyclobutanemeroterpenoids withpotent neuroprotective and anti-inflammatory activities from Hypericum beanii,
[0009] Chin.Chem.Lett.,2021,32,2338-2341.
[0010] [2]B.Hu,MYQian,JYZhang,XChou and L.Wu,Hyperhenrones:prenylatedα-pyrones with anti-inflammatory activity fromHypericum henryi,Phytochemistry,
[0011] 2024,220,114007. Summary of the Invention
[0012] The application aims to provide application of isoprenyl-alpha-pyrone dimer Hypericumonates D-F and pharmaceutically acceptable salts thereof in preparation of anti-neuroinflammatory drugs, which are extracted and separated from Hypericum for the first time.
[0013] To solve the technical problems of the application, the following technical solutions are provided.
[0014] An isoprenyl-alpha-pyrone dimer Hypericumonates D-F has a structural formula of D-F formula:
[0015]
[0016] The preparation method of the isoprenyl-alpha-pyrone dimer Hypericumonates D-F comprises the following steps:
[0017] Step A: 50 kg of dried Hypericum branches and leaves are extracted with 150-250 kg of methanol as a solvent at a temperature of 20-30 DEG C for 3-5 times, and each extraction time is 4 days; and the methanol is recovered by concentration under reduced pressure to obtain a crude extraction extract A;
[0018] Step B: the crude extraction extract A obtained in step A is subjected to solid phase extraction by using dichloromethane, ethyl acetate and methanol as extraction solvents to obtain three fractions: component A, component B and component C; component A is subjected to silica gel column chromatography by gradient elution with petroleum ether and dichloromethane in a volume ratio of 100:0-0:100; and different fractions are detected by using thin layer chromatography, and 7 fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6 and Fr.7 are obtained by combining according to the thin layer chromatography performance;
[0019] Step C: fraction Fr.2 is separated by using an RP-C18 chromatographic column by gradient elution with an eluent in a volume ratio of methanol to water of 40:60-100:0; and 5 sub-fractions Fr.2.A, Fr.2.B, Fr.2.C, Fr.2.D and Fr.2.E are obtained by combining according to the thin layer chromatography performance;
[0020] Step D: Fr.2.B was separated by silica gel column chromatography, eluted with a solvent gradient of petroleum ether / acetone (100:0-0:100, by volume) to give six sub-fractions: Fr.2.B.1, Fr.2.B.2, Fr.2.B.3, Fr.2.B.4, Fr.2.B.5 and Fr.2.B.6; the main component of Fr.2.B.4 was further purified by repeated silica gel column chromatography and gel column chromatography; finally, the main component was purified by semi-preparative high performance liquid chromatography, with acetonitrile and water (70:30, by volume) as the mobile phase at a flow rate of 3.0 mL / min to give Hypericumonate D and Hypericumonate E;
[0021] Step E: Fr.2.C was separated by silica gel column chromatography, eluted with a solvent gradient of petroleum ether and ethyl acetate (98:2-0:100, by volume) to give four sub-fractions: Fr.2.C.1, Fr.2.C.2, Fr.2.C.3 and Fr.2.C.4. Fr.2.C.2 was further purified by gel column chromatography with methanol as the solvent to give the main component Fr.2.C.2.B, which was further purified by semi-preparative high performance liquid chromatography, with acetonitrile and water (73:26, by volume) as the mobile phase at a flow rate of 3.0 mL / min to give the isoprenyl-α-pyrone dimer Hypericumonate F.
[0022] In step A, 100% methanol was used and cold maceration extraction was used.
[0023] In step B, the gradient elution concentrations of petroleum ether / dichloromethane were 100:0, 80:20, 70:30, 60:40, 50:50, 20:80 and 0:100, by volume.
[0024] In step C, the gradient elution concentrations of methanol / water were 40:60, 60:40, 75:25, 85:15 and 100:0, by volume.
[0025] In step D, the gradient elution concentrations of petroleum ether / acetone were 100:0, 80:20, 60:40, 50:50, 40:60 and 0:100, by volume.
[0026] In step E, the gradient elution concentrations of petroleum ether / ethyl acetate were 98:2, 90:10, 60:40 and 0:100, by volume. The semi-preparative high performance liquid chromatography used a reversed-phase octadecyl-bonded silica gel column.
[0027] The natural isoprenyl-α-pyrone dimer Hypericumonates D-F for use in the preparation of an anti-neuroinflammatory drug.
[0028] An anti-neuroinflammatory pharmaceutical composition comprising isoprenyl-α-pyrone dimer Hypericumonates D-F or a pharmaceutically acceptable salt.
[0029] An anti-neuroinflammatory pharmaceutical composition comprising isoprenyl-α-pyrone dimer Hypericumonates D-F or a pharmaceutically acceptable salt in a mass fraction of 0.1-99% and the rest is a pharmaceutical carrier or excipient. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1 The chemical structural formula of isoprenyl-α-pyrone dimer Hypericumonates D-F of the present application (the numbers in the formula are the carbon atom positions in the chemical structure) is as follows:
[0031] Figure 2 The nuclear magnetic resonance hydrogen spectrum of isoprenyl-α-pyrone dimer Hypericumonate D is as follows:
[0032] Figure 3 The nuclear magnetic resonance carbon spectrum of isoprenyl-α-pyrone dimer Hypericumonate D is as follows:
[0033] Figure 4 The nuclear magnetic resonance hydrogen spectrum of isoprenyl-α-pyrone dimer Hypericumonate E is as follows:
[0034] Figure 5 The nuclear magnetic resonance carbon spectrum of isoprenyl-α-pyrone dimer Hypericumonate E is as follows:
[0035] Figure 6 The nuclear magnetic resonance hydrogen spectrum of isoprenyl-α-pyrone dimer Hypericumonate F is as follows:
[0036] Figure 7 The nuclear magnetic resonance carbon spectrum of isoprenyl-α-pyrone dimer Hypericumonate F is as follows: DETAILED DESCRIPTION
[0037] Example 1
[0038] The chemical structural formula of isoprenyl-α-pyrone dimer Hypericumonates D-F of the present application (the numbers in the formula are the carbon atom positions in the chemical structure) is as follows:
[0039]
[0040] The preparation method of the isopentenyl-alpha-pyrone dimer Hypericumonates D-F, characterized in that it comprises the following steps:
[0041] Step A: 50 kg of dried Hypericum branches and leaves are extracted with 150-250 kg of methanol as solvent at a temperature of 20-30 DEG C for 3-5 times, and each extraction is performed for 4 days; the methanol is recovered by concentration under reduced pressure to obtain a crude extraction dreg A;
[0042] Step B: the crude extraction dreg A obtained in Step A is subjected to solid phase extraction using dichloromethane, ethyl acetate and methanol as extraction solvents to obtain three fractions: component A, component B and component C; component A is subjected to silica gel column chromatography with petroleum ether and dichloromethane as eluents in a volume ratio of 100:0-0:100; different fractions are detected by thin layer chromatography, and 7 fractions, Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6 and Fr.7, are obtained by combining fractions according to their thin layer chromatography performances;
[0043] Step C: Fr.2 is separated by using an RP-C18 chromatographic column, and eluents in a volume ratio of methanol to water of 40:60-100:0 are used for gradient elution; 5 sub-fractions, Fr.2.A, Fr.2.B, Fr.2.C, Fr.2.D and Fr.2.E, are obtained by combining according to their thin layer chromatography performances;
[0044] Step D: Fr.2.B is separated by silica gel column chromatography with petroleum ether / acetone as solvents in a volume ratio of 100:0-0:100 for gradient elution to obtain 6 sub-fractions, Fr.2.B.1, Fr.2.B.2, Fr.2.B.3, Fr.2.B.4, Fr.2.B.5 and Fr.2.B.6; the fraction Fr.2.B.4 is further purified by repeated silica gel column chromatography and gel chromatography to obtain the main component; finally, Hypericumonates D and Hypericumonates E are obtained by separating and purifying the main component by semi-preparative high performance liquid chromatography with acetonitrile and water in a volume ratio of 70:30 as eluents at a flow rate of 3.0 mL / min.
[0045] Step E: Fraction Fr.2.C was subjected to column chromatography on silica gel, eluted with petroleum ether and ethyl acetate in gradient of 98:2 to 0:100, to give four sub-fractions: Fr.2.C.1, Fr.2.C.2, Fr.2.C.3 and Fr.2.C.4. The main component Fr.2.C.2.B was obtained from Fr.2.C.2 by gel column chromatography, eluted with methanol. Fr.2.C.2.B was further purified by semi-preparative HPLC, acetonitrile and water in gradient of 73:26, at a flow rate of 3.0 mL / min, to give isoprenyl-α-pyrone dimer Hypericumonates F.
[0046] In step A, the methanol used was 100% methanol, and the extraction method was cold soak extraction.
[0047] In step B, the gradient elution concentration of petroleum ether / dichloromethane was in turn 100:0, 80:20, 70:30, 60:40, 50:50, 20:80 and 0:100.
[0048] In step C, the gradient elution concentration of methanol / water was in turn 40:60, 60:40, 75:25, 85:15 and 100:0.
[0049] In step D, the gradient elution concentration of petroleum ether / acetone was in turn 100:0, 80:20, 60:40, 50:50, 40:60 and 0:100.
[0050] In step E, the gradient elution concentration of petroleum ether / ethyl acetate was in turn 98:2, 90:10, 60:40 and 0:100; the semi-preparative HPLC column filler was reversed-phase octadecyl-bonded silica gel.
[0051] Use of a natural isoprenyl-α-pyrone dimer Hypericumonates D-F in the preparation of an anti-neuroinflammatory drug.
[0052] An anti-neuroinflammatory drug composition, comprising an isoprenyl-α-pyrone dimer Hypericumonates D-F or a pharmaceutically acceptable salt.
[0053] An anti-neuroinflammatory drug composition, comprising 0.1-99% of an isoprenyl-α-pyrone dimer Hypericumonates D-F or a pharmaceutically acceptable salt by mass fraction, and the rest being a pharmaceutical carrier or excipient.
[0054] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present application can be administered by any known administration method.
[0055] Example 2
[0056] Structure identification of isoprenyl-α-pyrone dimer Hypericumonates D-F: By routine identification methods, comprehensive analysis was carried out by high resolution mass spectrometry (HRESIMS), nuclear magnetic resonance spectrum (1H NMR, 13C NMR, 2D-NMR), infrared spectrum (IR), melting point (mp), optical rotation and other data, and its physicochemical properties are as follows: 1 H NMR, 13 C NMR, 2D-NMR), infrared spectrum (IR), melting point (mp), optical rotation and other data, and its physicochemical properties are as follows:
[0057] Hypericumonate D: colorless gum; (c 0.1, MeOH); UV (MeOH) λ max (logε) 280 (3.50) nm; IR (KBr) v max 2971.6, 2928.5, 1706.3, 1645.9, and 752.6 cm -1 ; HRESIMS m / z 475.2107 [M+H] + (calcd for C 29 H 31 O6, 475.2115). Nuclear magnetic resonance spectrum 1 H and 13 C NMR data are shown in Table 1.
[0058] Hypericumonate E: colorless gum; (c 0.1, MeOH); UV (MeOH) λ max (logε) 320 (3.85) nm; IR (KBr) v max 2963.0, 2927.1, 1696.2 and 1638.8 cm -1 ; HRESIMS m / z 497.1914 [M+Na] + (calcd for C 29 H 30 O6Na, 497.1935). Nuclear magnetic resonance spectrum 1 H and 13 C NMR data are shown in Table 1.
[0059] Hypericumonate F: colorless gum; (c 0.1, MeOH); UV (MeOH) λ max (logε) 330 (3.17) nm; IR (KBr) v max2964.5, 1689.1, 1262.5, 1097.3, 1031.1, 801.4 and 705.2 cm -1 HRESIMS m / z 547.2072 [M + Na] + ( calculated for C 33 H 32 O6Na, 547.2091). NMR spectra 1 H and 13 C NMR data are listed in Table 1.
[0060] Table 1: NMR data of Hypericumonates D-F in deuterated chloroform 1 H and 13 C NMR data (coupling constants J in Hz, chemical shifts δ in ppm).
[0061]
[0062] Example 3
[0063] To further verify the beneficial effects of the present application, the inhibitory effect of the isoprenyl-α-pyrone dimer Hypericumonates D-F prepared in Example 1 on NO in LPS-induced BV-2 cells was tested.
[0064] (1) Test sample:
[0065] Preparation of sample solution: an appropriate amount of Hypericumonates D-F was accurately weighed and dissolved in DMSO to prepare sample solutions of different concentrations for pharmacological activity testing.
[0066] Cell strain: mouse glial cells BV-2, preserved by the research group.
[0067] (2) Test method:
[0068] MTT method for determining cytotoxicity: Cells in logarithmic growth phase were digested and centrifuged, and the culture medium supernatant was discarded. The cells were resuspended in 10 mL complete culture medium, mixed evenly, and then 10 μL of the cell solution was taken to a counting plate under a microscope. The required cell solution and culture medium volume were calculated, and after the cell solution was mixed evenly, the cells were inoculated into a 96-well plate (BV-2 cells: 10,000 per well), and 100 μL of ddH2O was added to each well around the 96-well plate. Then it was placed in a 37°C, 5% CO2 incubator. After the cells were attached and grew for 24 hours, different concentrations of isoprenyl-α-pyrone dimer Hypericumonates D-F solution were added for treatment, with a final concentration of 10 or 20 μg / mL. After 24, 48, and 72 hours of cell treatment, 10 μL of MTT was added to each well, and after 4 hours of 37°C culture, it was centrifuged at 3000 rpm for 15 minutes, the culture medium supernatant was discarded, 160 μL of DMSO was added to each well, and it was placed in a 37°C constant temperature shaker for low-speed shaking for 20 minutes to fully dissolve the crystals. Then the absorbance value (OD value) of each well was measured at OD 490 nm on an enzyme-linked immunoassay instrument.
[0069]
[0070] NO concentration detection: Cells were collected and seeded in a 96-well plate (BV-2: 10,000 per well). After the cells were cultured for 12 hours, different concentrations of isoprenyl-α-pyrone dimer Hypericumonates D-F were prepared and added, with a final concentration of 10 μg / mL or 20 μg / mL, and a positive control of minocycline (MINO: 20 μg / mL) was treated. Incubate for 12 hours, prepare and add LPS to a final concentration of 5 μg / mL, and incubate the drug and LPS together for 24 hours. The culture medium supernatant was collected, centrifuged for 20 minutes, and the supernatant was taken. In the blank 96-well plate, 50 μL of standard and sample were added. In each well, 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added. The absorbance was measured at 540 nm wavelength using an enzyme-linked instrument, and the standard curve was plotted with the standard concentration on the X-axis and the absorbance on the Y-axis. The absorbance value was substituted into the regression equation to calculate the concentration of NO in the sample.
[0071] IC 50 Test, method same as above.
[0072] (3) Test results: According to the above test, under different concentrations (10 or 20 μM), the compounds Hypericumonates D-F all did not show cytotoxicity. By determining the inhibitory effect of Hypericumonates D-F on the NO in LPS-induced BV-2 cells, the results showed that, except for the compound Hypericumonates F, Hypericumonates D-E could significantly inhibit the concentration of NO in LPS-induced BV-2 cells, and the IC 50 values were 2.68 ± 0.76 and 2.41 ± 0.31, respectively, and the activity was obviously stronger than that of the positive control drug (minocycline, IC 50 = 19.09 ± 1.34), as shown in Table 2.
[0073] Table 2 Inhibitory activity of Hypericumonates D-F on the concentration of NO in LPS-induced BV-2 cells
[0074]
[0075]
[0076] (4) Test conclusion: The compounds Hypericumonates D-E have significant inhibitory activity on the concentration of NO in LPS-induced BV-2 cells, show obvious anti-inflammatory effect, and the activity is stronger than that of the positive control drug minocycline, therefore, the Hypericumonates D-E of the present application can be used for preparing anti-neuroinflammatory drugs.
[0077] The above only describes the preferred embodiments of the present application, and it should be noted that, for the researchers in the technical field, any other improvement and modification made without departing from the method and content of the present application should be considered as the protection scope of the present application.
Claims
1. A method for producing a isoprenyl- pyranone dimer Hypericumonate E of the formula E α Comprising the following steps: Step A: 50 kg of dried Hypericum branches and leaves were extracted with 150-250 kg of methanol as solvent at a temperature of 20-30 °C for 3-5 times, and each extraction lasted for 4 days. The methanol was recovered by concentration under reduced pressure to obtain a crude extract A; Step B: The crude extract A obtained in step A was subjected to solid phase extraction using dichloromethane, ethyl acetate and methanol as extraction solvents to obtain 3 fractions: component A, component B and component C. Component A was subjected to silica gel column chromatography with petroleum ether and dichloromethane in a volume ratio of 100:0-0:100 for gradient elution, and then combined according to the thin layer chromatography performance to obtain 7 fractions: Fr. 1, Fr. 2, Fr. 3, Fr. 4, Fr. 5, Fr. 6 and Fr. 7; Step C: Fraction Fr. 2 was separated by RP-C18 column chromatography with a gradient elution of eluent in a volume ratio of methanol to water of 40:60-100:0, and combined according to the thin layer chromatography performance to obtain 5 sub-fractions: Fr.2.A, Fr.2.B, Fr.2.C, Fr.2.D and Fr.2.E; Step D: Fr.2.B was separated by silica gel column chromatography with a gradient elution of solvent in a volume ratio of petroleum ether to acetone of 100:0-0:100 to obtain 6 sub-fractions: Fr.2.B.1, Fr.2.B.2, Fr.2.B.3, Fr.2.B.4, Fr.2.B.5 and Fr.2.B.
6. Fraction Fr.2.B.4 was further purified by repeated silica gel column chromatography and gel column chromatography to obtain the main component. The main component was separated and purified by semi-preparative high performance liquid chromatography with a volume ratio of acetonitrile to water of 70:30 and a flow rate of 3.0 mL / min to obtain Hypericumonate E. 。 2. The method for preparing Hypericumonate E according to claim 1, characterized in that, In step A, the methanol is 100% industrial methanol, and the extraction method is cold extraction.
3. The method of preparing the prenyl-α-pyrone dimer Hypericumonate E according to claim 1, characterized in that, In step B, the gradient elution concentration of petroleum ether / dichloromethane is in a volume ratio of 100:0, 80:20, 70:30, 60:40, 50:50, 20:80 and 0:100, respectively.
4. The method of preparing the prenyl-α-pyrone dimer Hypericumonate E according to claim 1, characterized in that, In step C, the gradient elution concentration of methanol / water is in a volume ratio of 40:60, 60:40, 75:25, 85:15 and 100:0, respectively.
5. The method of preparing the prenyl-α-pyrone dimer Hypericumonate E according to claim 1, characterized in that, In step D, the gradient elution concentration of petroleum ether / acetone is in a volume ratio of 100:0, 80:20, 60:40, 50:50, 40:60 and 0:100, respectively.