Isopentenyl-alpha-pyrone dimer Hypericumonates D-F as well as preparation method and application of isopentenyl-alpha-pyrone dimer Hypericumonates D-F

Abstract

The invention relates to an isopentenyl-alpha-pyrone dimer Hyperumonates D-F as well as a preparation method and application thereof, and belongs to the field of medical chemistry, the isopentenyl-alpha-pyrone dimer Hyperumonates D-F is obtained by extracting and separating from leaves and branches of Hypericum monogynum for the first time, and the structural formula of the isopentenyl-alpha-pyrone dimer Hyperumonates D-F is D-F. The invention further discloses a preparation method of the isopentenyl-alpha-pyrone dimer Hyperumonates D-F. The isopentenyl-alpha-pyrone dimer Hypericumonates D-F has the advantages that the compounds D-E have remarkable inhibition effects on generation of NO in mouse microglial cells (BV-2 cells) induced by lipopolysaccharide (LPS), the activity of the compounds D-E is higher than that of positive medicine minocycline, and the compounds D-E have application potentials in the field of neuroinflammation medicines, and the invention also relates to a pharmaceutical composition which comprises the isopentenyl-alpha-pyrone dimer Hypericumonates D-F and can be used for preparing medicines for treating neuroinflammation. And pharmaceutically acceptable auxiliary materials, wherein the pharmaceutical composition has the effect of resisting neuroinflammation.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and specifically to a natural medicine Hypericumonates DF, which is an isopentenyl-α-pyrone dimer with anti-neuroinflammatory activity and is extracted and separated from Hypericum perforatum branches and leaves, as well as a preparation method and application thereof in the treatment of neuroinflammation. Background Art

[0002] Neuroinflammation is a disease observed in the central nervous system (CNS) under the stimulation of infection, toxic metabolites, trauma or autoimmunity. During the stimulation process, glial cells such as microglia and astrocytes are activated, releasing a large number of inflammatory mediators, leading to abnormal reactions or damage to neural tissue. It has a certain protective effect, but excessive neuroinflammation can cause a variety of 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 during use. Therefore, it is very important to discover new anti-neuroinflammatory drug candidates. Natural medicines have an irreplaceable and important position in innovative drug discovery. Their unique novel structure and good biological activity will continue to play a key role in the future field of drug research and development. Therefore, searching and discovering anti-neuroinflammatory drug candidates from natural medicines has become an important way in this research field.

[0003] Isopentenyl-α-pyrone dimer is a parent nucleus formed by a [2+2] cyclization reaction of two molecules of α-pyrone and dimethylpyran ring through an intracyclic double bond. Due to its unique chemical structure and good biological activity, it has broad prospects for development and application in the field of medicine. The isopentenyl-α-pyrone dimer Hypericumonates D-F described in the present invention is obtained by a [2+2] cyclization reaction between a double bond within a pyran ring of one molecule and a double bond within an α-pyrone ring of another molecule, and the two molecules are connected by cyclobutane to form a 6 / 6 / 4 / 6 / 6 ring system parent nucleus. This type of compound has not been reported in Hypericum, and so far, only four pairs of isopentenyl-α-pyrone dimers have been reported from Hypericum plants (see references 1 and 2 for related studies). This type of compound has 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 planted in central and southern provinces of my country as a landscape plant and folk medicinal plant. According to the Dictionary of Chinese Materia Medica, its fruit can be used as a substitute for Forsythia suspensa, and its root can eliminate rheumatism, relieve cough, and treat traumatic injuries. At present, the research on the chemical components of Hypericum monogynum is mainly focused on compounds such as phloroglucinol, spironolactone, flavonoids, and triterpenes. Therefore, it is of great research value and significance to explore novel isopentenyl-α-pyrone dimers with anti-neuroinflammatory activity from Hypericum monogynum.

[0005] References:

[0006] [1] B. Zhen, X. Y. Suo, J. Dang, H. L. Yu, Y. D. Tao, J. J. Wang, L. Li, M. B. Lin, Q.

[0007] Hou, WPWang,

[0010] [2] B.Hu, MYQian, JYZhang, Summary of the invention

[0012] The object of the present invention is to provide an application of an isopentenyl-α-pyrone dimer Hypericumonates DF extracted and separated from Hypericum perforatum for the first time and a pharmaceutically acceptable salt thereof in the preparation of an anti-neuroinflammatory drug.

[0013] In order to solve the technical problem of the present invention, the following technical solution is provided:

[0014] An isopentenyl-α-pyrone dimer Hypericumonates DF, whose structural formula is D to F:

[0015]

[0016] The preparation method of the isopentenyl-α-pyrone dimer Hypericumonates DF is characterized by comprising the following steps:

[0017] Step A: taking 50 kg of dried Hypericum perforatum branches and leaves, using 150-250 kg of methanol as solvent, extracting 3-5 times at a temperature of 20-30° C., each extraction time being 4 days, and concentrating under reduced pressure to recover methanol to obtain a crude extract A;

[0018] Step B: using dichloromethane, ethyl acetate and methanol as extraction solvents, the crude extract A obtained in step A is subjected to solid phase extraction to obtain three fractions: component A, component B, and component C; component A is subjected to silica gel column chromatography, and gradient elution is performed with petroleum ether and dichloromethane in a volume ratio of 100:0-0:100; different fractions are then detected using thin layer chromatography technology, and seven fractions are combined according to their thin layer chromatography performance to obtain: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7;

[0019] Step C: Separate the fraction Fr.2 using an RP-C18 column, perform gradient elution with an eluent having a volume ratio of methanol to water of 40:60 to 100:0, and combine the fractions according to the thin layer chromatography performance to obtain five sub-fractions: Fr.2.A, Fr.2.B, Fr.2.C, Fr.2.D and Fr.2.E;

[0020] Step D: Fr.2.B was separated by silica gel column chromatography, and eluted with petroleum ether / acetone volume ratio of 100:0-0:100 as solvent gradient 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 purified by repeated silica gel column chromatography and gel chromatography column to obtain the main component; finally, the main component was separated and purified by semi-preparative high performance liquid phase separation, with acetonitrile and water volume ratio of 70:30 and flow rate of 3.0 mL / min to obtain Hypericumonate D and Hypericumonate E;

[0021] Step E: Fraction Fr.2.C was chromatographed on a silica gel column using a gradient elution of petroleum ether and ethyl acetate in a volume ratio of 98:2 to 0:100 to obtain four subfractions: Fr.2.C.1, Fr.2.C.2, Fr.2.C.3 and Fr.2.C.4. Component Fr.2.C.2 was eluted on a gel chromatography column using methanol as solvent to obtain the main component Fr.2.C.2.B, which was further purified by semi-preparative high performance liquid chromatography using an acetonitrile to water volume ratio of 73:26 at a flow rate of 3.0 mL / min to obtain isopentenyl-α-pyrone dimer Hypericumonate F.

[0022] In step A, the methanol used is 100% methanol, and the extraction method is cold soaking extraction.

[0023] In step B, the gradient elution concentrations of petroleum ether / dichloromethane are 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 are volume ratios of 40:60, 60:40, 75:25, 85:15, and 100:0, respectively.

[0025] In step D, the gradient elution concentrations of petroleum ether / acetone are 100:0, 80:20, 60:40, 50:50, 40:60, and 0:100 by volume.

[0026] In step E, the gradient elution of petroleum ether / ethyl acetate is carried out in the following volume ratios: 98:2, 90:10, 60:40, and 0:100. The semi-preparative high performance liquid chromatography column filler is reversed-phase octadecyl bonded silica gel.

[0027] Application of a natural isopentenyl-α-pyrone dimer Hypericumonates DF in the preparation of anti-neuroinflammatory drugs.

[0028] An anti-neuroinflammatory pharmaceutical composition comprises isopentenyl-α-pyrone dimer Hypericumonates DF or a pharmaceutically acceptable salt thereof.

[0029] An anti-neuroinflammation pharmaceutical composition contains 0.1-99% by mass of isopentenyl-α-pyrone dimer Hypericumonates DF or a pharmaceutically acceptable salt thereof, and the rest is a pharmaceutical carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The structural formula of the isopentenyl-α-pyrone dimer Hypericumonates DF of the present invention;

[0031] Figure 2 This is the H NMR spectrum of isopentenyl-α-pyrone dimer Hypericumonate D;

[0032] Figure 3 This is the carbon NMR spectrum of isopentenyl-α-pyrone dimer Hypericumonate D;

[0033] Figure 4 is the H NMR spectrum of isopentenyl-α-pyrone dimer Hypericumonate E;

[0034] Figure 5 This is the carbon NMR spectrum of isopentenyl-α-pyrone dimer Hypericumonate E;

[0035] Figure 6 This is the H NMR spectrum of isopentenyl-α-pyrone dimer Hypericumonate F;

[0036] Figure 7 This is the carbon NMR spectrum of isopentenyl-α-pyrone dimer Hypericumonate F; DETAILED DESCRIPTION

[0037] Example 1

[0038] The chemical structural formula of the isopentenyl-α-pyrone dimer Hypericumonates DF referred to in the examples (the numbers in the formula are the positions of carbon atoms in the chemical structure) is as follows:

[0039]

[0040] The preparation method of the isopentenyl-α-pyrone dimer Hypericumonates DF is characterized by comprising the following steps:

[0041] Step A: taking 50 kg of dried Hypericum perforatum branches and leaves, using 150-250 kg of methanol as solvent, extracting 3-5 times at a temperature of 20-30° C., each extraction time being 4 days, and concentrating under reduced pressure to recover methanol to obtain a crude extract A;

[0042] Step B: using dichloromethane, ethyl acetate and methanol as extraction solvents, the crude extract A obtained in step A is subjected to solid phase extraction to obtain three fractions: component A, component B, and component C; component A is subjected to silica gel column chromatography, and gradient elution is performed with petroleum ether and dichloromethane in a volume ratio of 100:0-0:100; different fractions are then detected using thin layer chromatography technology, and seven fractions are combined according to their thin layer chromatography performance to obtain: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7;

[0043] Step C: Separate the fraction Fr.2 using an RP-C18 column, perform gradient elution with an eluent having a volume ratio of methanol to water of 40:60 to 100:0, and combine the fractions according to the thin layer chromatography performance to obtain five sub-fractions: Fr.2.A, Fr.2.B, Fr.2.C, Fr.2.D and Fr.2.E;

[0044] Step D: Fr.2.B was separated by silica gel column chromatography, and eluted with a solvent gradient of petroleum ether / acetone volume ratio 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 purified by repeated silica gel column chromatography and gel chromatography to obtain the main component. Finally, the main component was separated and purified by semi-preparative high performance liquid phase separation, with an acetonitrile and water volume ratio of 70:30 and a flow rate of 3.0 mL / min to obtain Hypericumonates D and Hypericumonates E.

[0045] Step E: Fraction Fr.2.C was chromatographed on a silica gel column using a gradient elution with petroleum ether and ethyl acetate in a volume ratio of 98:2 to 0:100 to obtain four subfractions: Fr.2.C.1, Fr.2.C.2, Fr.2.C.3 and Fr.2.C.4. Component Fr.2.C.2 was eluted with methanol as solvent to obtain the main component Fr.2.C.2.B. Fr.2.C.2.B was further purified by semi-preparative high performance liquid chromatography with an acetonitrile to water volume ratio of 73:26 and a flow rate of 3.0 mL / min to obtain isopentenyl-α-pyrone dimer Hypericumonates F.

[0046] In step A, the methanol used is 100% methanol, and the extraction method is cold soaking extraction.

[0047] In step B, the gradient elution concentration of petroleum ether / dichloromethane is 100:0, 80:20, 70:30, 60:40, 50:50, 20:80, and 0:100 by volume.

[0048] In step C, the gradient elution concentrations of methanol / water are volume ratios of 40:60, 60:40, 75:25, 85:15, and 100:0, respectively.

[0049] In step D, the gradient elution using petroleum ether / acetone has a volume ratio of 100:0, 80:20, 60:40, 50:50, 40:60, and 0:100.

[0050] In step E, the gradient elution of petroleum ether / ethyl acetate is carried out in the following volume ratios: 98:2, 90:10, 60:40, and 0:100; the semi-preparative high performance liquid chromatography column filler is reversed-phase octadecyl bonded silica gel.

[0051] Application of a natural isopentenyl-α-pyrone dimer Hypericumonates DF in the preparation of anti-neuroinflammatory drugs.

[0052] An anti-neuroinflammatory pharmaceutical composition comprises isopentenyl-α-pyrone dimer Hypericumonates DF or a pharmaceutically acceptable salt thereof.

[0053] An anti-neuroinflammation pharmaceutical composition contains 0.1-99% by mass of isopentenyl-α-pyrone dimer Hypericumonates DF or a pharmaceutically acceptable salt thereof, and the rest is a pharmaceutical carrier or excipient.

[0054] In order to achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

[0055] Example 2

[0056] The structure of isopentenyl-α-pyrone dimer Hypericumonates DF was identified by conventional identification methods, high resolution mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR, 2D-NMR), infrared spectrum (IR), melting point (mp), optical rotation and other data were comprehensively analyzed, and its physical and chemical properties are as follows:

[0057] Hypericumonate D: colorless colloid; (c 0.1,MeOH); UV(MeOH)λ max (logε)280(3.50)nm; IR(KBr)ν max 2971.6, 2928.5, 1706.3, 1645.9, and 752.6cm -1; HRESIMS m / z475.2107[M+H] + (The calculated value is C 29 H 31 O 6 ,475.2115). NMR spectrum 1 H and 13 C NMR data, see Table 1.

[0058] Hypericumonate E: colorless colloid; (c 0.1,MeOH); UV(MeOH)λ max (logε)320(3.85)nm; IR(KBr)ν max 2963.0, 2927.1, 1696.2 and 1638.8cm -1 ; HRESIMS m / z 497.1914[M+Na] + (The calculated value is C 29 H 30 O 6 Na, 497.1935). NMR spectrum 1 H and 13 CNMR data, see Table 1.

[0059] Hypericumonate F: colorless colloid; (c 0.1,MeOH); UV(MeOH)λ max (logε)330(3.17)nm; IR(KBr)ν max 2964.5, 1689.1, 1262.5, 1097.3, 1031.1, 801.4 and 705.2 cm -1 ; HRESIMS m / z 547.2072[M+Na] + (The calculated value is C 33 H 32 O 6 Na, 547.2091). NMR spectrum 1 H and 13 C NMR data, see Table 1.

[0060] Table 1: Compound DF in deuterated chloroform 1 H and 13 C NMR data (coupling constant J in Hz, chemical shift δ in ppm).

[0061]

[0062] Example 3

[0063] In order to further verify the beneficial effects of the present invention, the isopentenyl-α-pyrone dimer Hypericumonates DF prepared in Example 1 was used to inhibit NO in LPS-induced BV-2 cells;

[0064] (1) Test samples:

[0065] Preparation of sample solution: Accurately weigh an appropriate amount of Hypericumonates DF and use DMSO to prepare sample solutions of different concentrations for pharmacological activity testing.

[0066] Cell line: Mouse glial cell line BV-2, retained by this research group.

[0067] (2) Test method:

[0068] ① MTT method to determine cytotoxicity: Take cells in the logarithmic growth phase, digest and centrifuge, discard the culture medium supernatant, resuspend the cells with 10mL complete culture medium, mix well, take 10μL of cell solution to the counting plate, and count under a microscope. Calculate the required volume of cell solution and culture medium, prepare the cell solution and mix well, then inoculate the cells in a 96-well plate (BV-2 cells: 10,000 / well), and add 100μL ddH2O to each well around the 96-well plate. 2 O. Then place in 37°C, 5% CO 2 In a constant temperature incubator. After the cells were attached and grown for 24 hours, different concentrations of isopentenyl-α-pyrone dimer Hypericumonates DF solution were added to make the final concentration: 10 or 20μg / mL. After the cells were treated with drugs for 24, 48 and 72 hours, 10μL MTT was added to each well, and after culturing at 37℃ for 4 hours, centrifuged at 3000rpm for 15 minutes, the supernatant of the culture medium was aspirated and discarded, 160μL DMSO was added to each well, and the cells were placed on a 37℃ constant temperature shaker and shaken at a low speed for 20 minutes to fully dissolve the crystals, and then the absorbance value (OD value) of each well was measured at OD 490nm on an enzyme-linked immunosorbent assay.

[0069]

[0070] ② Detection of nitric oxide (NO) concentration: Collect cells and plate them in 96-well plates (BV-2: 10,000 cells / well). After 12 hours of cell culture, prepare and add different concentrations of isopentenyl-α-pyrone dimer Hypericumonates DF to a final concentration of 10μg / mL or 20μg / mL, as well as the positive control minocycline (MINO: 20μg / mL). Incubate for 12 hours, prepare and add LPS to a final concentration of 5μg / mL, and incubate the drug with LPS for 24 hours. Collect the supernatant of the culture medium, centrifuge for 20 minutes, and take the supernatant. Add the prepared standards and samples to a blank 96-well plate at 50μL / well. Add 50μL Griess Reagent I and 50μL Griess Reagent II to each well. The absorbance was measured at a wavelength of 540 nm using an enzyme marker, and a standard curve was drawn with the X-axis as the standard concentration and the Y-axis as the absorbance. The absorbance value was substituted into the regression equation to calculate the concentration of NO in the sample.

[0071] ③IC 50 Test, the method is the same as above.

[0072] (3) Experimental results: According to the above test, at different concentrations (10 or 20 μM), the compounds Hypericumonates D-F showed no cytotoxicity. By measuring the inhibitory effect of Hypericumonates DF on NO in LPS-induced BV-2 cells, the results showed that, except for the compound Hypericumonates F, Hypericumonates DE could significantly inhibit the NO concentration in LPS-induced BV-2 cells, and its IC 50 The values ​​were 2.68±0.76 and 2.41±0.31, respectively, and the activity was significantly 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 DF on NO concentration in BV-2 cells induced by LPS

[0074]

[0075]

[0076] (4) Experimental conclusion: The compound Hypericumonates DE has significant inhibitory activity on the NO concentration in LPS-induced BV-2 cells, showing obvious anti-inflammatory effects, and its activity is stronger than that of the positive control drug minocycline. Therefore, Hypericumonates DE of the present invention can be used to prepare anti-neuroinflammatory drugs.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for researchers in this technical field, any other improvements and modifications made without departing from the method and content of the present invention should also be regarded as within the scope of protection of the present invention.

Claims

1. An isopentenyl-α-pyrone dimer Hypericumonates DF, having a structural formula of D to F 2. A method for preparing the isopentenyl-α-pyrone dimer Hypericumonates DF as claimed in claim 1, characterized in that: The following steps are involved: Step A: taking 50 kg of dried Hypericum perforatum branches and leaves, using 150-250 kg of methanol as solvent, extracting 3-5 times at a temperature of 20-30° C., each extraction time being 4 days, and concentrating under reduced pressure to recover methanol to obtain a crude extract A; Step B: using dichloromethane, ethyl acetate and methanol as extraction solvents, the crude extract A obtained in step A is subjected to solid phase extraction to obtain three fractions: component A, component B, and component C; component A is subjected to silica gel column chromatography, and gradient elution with petroleum ether and dichloromethane in a volume ratio of 100:0-0:100 is performed, and then seven fractions are combined according to their thin layer chromatography performance to obtain: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7; Step C: Separate the fraction Fr.2 using an RP-C18 column, perform gradient elution with an eluent having a volume ratio of methanol to water of 40:60 to 100:0, and combine the fractions according to the thin layer chromatography performance to obtain five 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, and eluted with petroleum ether / acetone volume ratio of 100:0-0:100 as solvent gradient 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 purified by repeated silica gel column chromatography and gel chromatography column to obtain the main component, and the main component was separated and purified by semi-preparative high performance liquid phase separation, with acetonitrile and water volume ratio of 70:30 and flow rate of 3.0 mL / min to obtain Hypericumonate D and Hypericumonate E; Step E: Fraction Fr.2.C was chromatographed on a silica gel column using a gradient elution with petroleum ether and ethyl acetate in a volume ratio of 98:2 to 0:100 to obtain four subfractions: Fr.2.C.1, Fr.2.C.2, Fr.2.C.3 and Fr.2.C.

4. Component Fr.2.C.2 was eluted with methanol as solvent to obtain the main component Fr.2.C.2.B. Fr.2.C.2.B was further purified by semi-preparative high performance liquid chromatography with an acetonitrile to water volume ratio of 73:26 at a flow rate of 3.0 mL / min to obtain the isopentenyl-α-pyrone dimer Hypericumonate F.

3. The method for preparing an isopentenyl-α-pyrone dimer Hypericumonates DF according to claim 2, characterized in that: In step A, the methanol is 100% industrial methanol, and the extraction method is cold soaking extraction.

4. The method for preparing the isopentenyl-α-pyrone dimer Hypericumonates DF according to claim 2, characterized in that: In step B, the gradient elution concentration of petroleum ether / dichloromethane is 100:0, 80:20, 70:30, 60:40, 50:50, 20:80, and 0:100 by volume.

5. The method for preparing the isopentenyl-α-pyrone dimer Hypericumonates DF according to claim 2, characterized in that: In step C, the gradient elution using methanol / water has a volume ratio of 40:60, 60:40, 75:25, 85:15, and 100:

0.

6. The method for preparing the isopentenyl-α-pyrone dimer Hypericumonates DF according to claim 2, characterized in that: In step D, the gradient elution using petroleum ether / acetone has a volume ratio of 100:0, 80:20, 60:40, 50:50, 40:60, and 0:

100.

7. The method for preparing the isopentenyl-α-pyrone dimer Hypericumonates DF according to claim 2, characterized in that: In step E, a gradient elution of petroleum ether / ethyl acetate with a volume ratio of 98:2, 90:10, 60:40, and 0:100 is used, and the semi-preparative high performance liquid chromatography column filler is reversed-phase octadecyl bonded silica gel.

8. Use of the isopentenyl-α-pyrone dimer Hypericumonates DF as claimed in claim 1 in the preparation of anti-neuroinflammatory drugs.

9. An anti-neuroinflammatory pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the isopentenyl-α-pyrone dimer Hypericumonates DF or a pharmaceutically acceptable salt thereof according to claim 1.

10. An anti-neuroinflammatory pharmaceutical composition according to claim 9, characterized in that: The pharmaceutical composition contains 0.1-99% by mass of the isopentenyl-α-pyrone dimer Hypericumonates DF or a pharmaceutically acceptable salt thereof as claimed in claim 1, and the rest is a pharmaceutical carrier or excipient.

Images