Polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity as well as preparation method and application of polycyclic polyisopentenyl phloroglucinol compound

Abstract

The invention discloses a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity as well as a preparation method and application thereof, and belongs to the technical field of medicinal compounds. The invention discloses a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity. The molecular formula of the polycyclic polyisopentenyl phloroglucinol compound is C33H40O5. The compound is reported for the first time, and the anti-inflammatory activity of the compound on lipopolysaccharide (LPS) induced mouse mononuclear macrophage leukemia cells (RAW264.7) is evaluated. The result shows that the compound can inhibit the generation of an inflammatory medium nitric oxide (NO) in LPS-induced RAW264.7 cells, so that the compound can be used as an anti-inflammatory drug.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and more particularly to a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity, a preparation method and application thereof. Background Art

[0002] Polycyclic polyisopentenyl phloroglucinol derivatives (PPAPs) are a class of natural products with novel structures and high synthetic difficulty. They have a core derived from highly oxidized and various acyl phloroglucinols, and their side chains have multiple isopentenyl or geranyl groups. These compounds are widely found in plants of the genus Hypericum in the family Garcinia family and have a variety of pharmacological effects such as antibacterial, antidepressant, antiviral, and antitumor. PPAPs have attracted extensive attention from researchers due to their complex structure and significant biological activity.

[0003] As a member of the genus Hypericum in the family Garcinia, the research on the chemical composition and pharmacological activity of Hypericum henryi H.Lév.&Vaniot has always been the focus of research. It is widely distributed in southwest my country. Traditionally, the plant has been used to treat a variety of diseases, such as antidepressant, antibacterial hemostasis, liver protection, and treatment of gastrointestinal diseases.

[0004] Inflammation is the first response of the immune system to infection or stimulation, and macrophages play a vital role in the inflammatory process. In the presence of stimuli such as lipopolysaccharide (LPS), activated macrophages produce various cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). In traditional medicine, steroids and non-steroidal anti-inflammatory drugs have only limited therapeutic effects on inflammation. Moreover, these drugs can also produce serious side effects, such as gastrointestinal disorders, kidney damage, skin reactions, etc. Therefore, it is necessary to find new, effective and safe anti-inflammatory drugs. Natural products are a valuable source for the development of new drugs because of their diversity, multi-targets and low toxicity and side effects. They may improve the potential adverse reactions of current anti-inflammatory drugs and become new conventional drugs as well as complementary and alternative drugs.

[0005] Therefore, providing a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity and a preparation method and application thereof is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity, and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity, the molecular structure of which is as follows:

[0009]

[0010] Furthermore, the preparation method of the polycyclic polyisopentenyl phloroglucinol compound having anti-inflammatory activity comprises the following specific steps:

[0011] (1) The air-dried aerial part of Hypericum cyrtonema is made into powder, and the powder is extracted with dichloromethane at room temperature three times, each time for two days; the extract is concentrated and dried by a rotary evaporator to obtain a dichloromethane total extract; the mass volume ratio of the powder to the dichloromethane is 1:5;

[0012] (2) The total dichloromethane extract obtained in step (1) was mixed with 200 g of 100-200 mesh silica gel, and 600 g of 200-300 mesh silica gel was used for column chromatography separation, and petroleum ether-ethyl acetate was used as the mobile phase for gradient elution, and the volume ratio of petroleum ether-ethyl acetate was 1:0, 50:1, 30:1, 15:1, 8:1, 4:1, 1.5:1, and 1:2; the eluate was collected and concentrated to dryness, and analyzed by thin layer chromatography, and similar components were combined to finally obtain 6 components Fr.A-Fr.F;

[0013] (3) The Fr.D obtained in step (2) is subjected to a macroporous adsorption resin chromatography column and gradient eluted using 30%, 50%, 70% methanol aqueous solution and 100% methanol as mobile phases; the eluate is collected, analyzed by thin layer chromatography, and combined into Fr.D.1-Fr.D.6;

[0014] (4) Fr.D.4 obtained in step (3) was eluted by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 10:1, 8:1, 4:1, 2:1, and 1:10 as the mobile phase, and the same components were combined to obtain Fr.D.4.1-Fr.D.4.5; Fr.D.4.2 was then eluted on a Sephadex LH-20 gel column using an eluent of dichloromethane:methanol = 1:1 to obtain five components Fr.D.4.2.1-Fr.D.4.2.5;

[0015] (5) Fr.D.4.2.2 obtained in step (4) was purified by semi-preparative liquid chromatography using 70% methanol aqueous solution as the mobile phase to obtain compound I; the flow rate was 10 mL / min.

[0016] Furthermore, the polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity is used in the preparation of anti-inflammatory drugs.

[0017] The total dichloromethane extract was purified by silica gel column chromatography, macroporous adsorption resin chromatography column, hydroxypropyl dextran gel (Sephadex LH-20) chromatography column and semi-preparative liquid chromatography to obtain compound I.

[0018] The polycyclic polyisopentenyl phloroglucinol compounds with anti-inflammatory activity were analyzed by HR-ESI-MS, 1 H NMR, 13 C NMR, HSQC, HMBC, 1 H- 1 The samples were identified by H COSY spectroscopy.

[0019] The invention discloses an application of a polycyclic polyisopentenyl phloroglucinol compound in the preparation of an anti-inflammatory drug. The compound has a potential inhibitory effect on the production of NO by LPS-stimulated RAW264.7 cells.

[0020] The structure, preparation method and pharmacological activity of the new compound described in the present invention have not been reported in the literature.

[0021] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity and its preparation method and application, and proves that the polycyclic polyisopentenyl phloroglucinol compound has significant anti-inflammatory activity by evaluating its inhibitory effect on the secretion of inflammatory mediator NO in RAW264.7 cells stimulated by LPS. In the field of pharmaceutical compounds, the natural product can be used as a lead compound to develop anti-inflammatory drugs with new mechanisms of action and new targets, providing new solutions for the treatment of inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 is the HR-ESI-MS chart of compound Ⅰ of the present invention;

[0024] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of compound Ⅰ of the present invention;

[0025] Figure 3 is the carbon nuclear magnetic resonance spectrum of compound Ⅰ of the present invention;

[0026] Figure 4 The main HMBC and 1H- 1 H COSY diagram;

[0027] Figure 5 is the structural formula of compound Ⅰ of the present invention;

[0028] Figure 6 is the structural formula of compound Ⅰ of the present invention with atomic numbers;

[0029] Figure 7 The figure shows the effects of the compound I of the present invention and positive drugs on the secretion of NO in RAW264.7 cells stimulated by LPS. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] The preparation method of polycyclic polyisopentenyl phloroglucinol compounds comprises the following specific steps:

[0033] (1) The air-dried aerial part of Hypericum cyrtonema (10.0 kg) was powdered and extracted with dichloromethane (50.0 L each time) at room temperature for three times, each time for two days. The extract was concentrated and dried by rotary evaporator to obtain a dichloromethane total extract (367.0 g).

[0034] (2) The total dichloromethane extract obtained in step (1) was mixed with 200 g of 100-200 mesh silica gel (for mixing samples), and 600 g of 200-300 mesh silica gel (for column loading) was used for column chromatography separation, and petroleum ether-ethyl acetate (1:0, 50:1, 30:1, 15:1, 8:1, 4:1, 1.5:1, 1:2, v / v) was used as the mobile phase for gradient elution. The eluate was collected and concentrated to dryness, analyzed by thin layer chromatography (TLC) (using vanillin concentrated sulfuric acid solution as a color developer, heating for color development), and similar components were combined to finally obtain 6 components (Fr.A-Fr.F).

[0035] (3) Fr.D (5.8 g) obtained in step (2) was separated by a macroporous adsorption resin column, and gradient elution was performed using 30%, 50%, 70% methanol aqueous solution and 100% methanol as mobile phases. The eluate was collected and analyzed by TLC (using vanillin concentrated sulfuric acid solution as a color developer, heating for color development), and combined into Fr.D.1-Fr.D.6.

[0036] (4) Fr.D.4 (1.6 g) obtained in step (3) was eluted by silica gel column chromatography using petroleum ether:ethyl acetate volume ratios of 10:1, 8:1, 4:1, 2:1, and 1:10 as mobile phases, and the same fractions were combined to obtain Fr.D.4.1-Fr.D.4.5. Fr.D.4.2 (167.2 mg) was then eluted on a Sephadex LH-20 gel column using an eluent of dichloromethane:methanol = 1:1 to obtain five fractions Fr.D.4.2.1-Fr.D.4.2.5.

[0037] (5) Fr.D.4.2.2 (30.7 mg) obtained in step (4) was purified by semi-preparative liquid chromatography using methanol and water (70:30, 10 mL / min) as the mobile phase to obtain compound I (7.8 mg, t R =18.1min).

[0038] Example 2 Structural Identification of New Compounds

[0039] Using HRESIMS, 1 H NMR, 13 C NMR, HSQC, HMBC, 1 H- 1 The structure of the new compound Ⅰ obtained in step (5) was identified by spectral techniques such as H COSY.

[0040] Compound Ⅰ, colorless oil. Figure 1 As shown, according to HR-ESI-MS data (m / z 517.2929[M+H] + ; C 33 H 41 O 5 Calculated value 517.2949) and 13 C NMR data (Table 1), the molecular formula is inferred to be C 33 H 40 O 5 . H NMR spectrum ( Figure 2 ) shows the presence of a monosubstituted benzene [δ H 7.79 (2H, dd, J = 8.2, 1.4 Hz), 7.41 (2H, td, J = 7.9, 7.3, 1.4 Hz), 7.53 (1H, m)], four olefin protons (δ H 5.01, 5.10, 5.58 and 5.58) and seven methyl groups (δ H 1.28-1.70). NMR carbon spectrum ( Figure 3 ) shows the presence of 33 carbons, including 7 quaternary carbons (δ C 42.5 and δ C 65.2. A non-conjugated carbonyl group δC 207.3, a 1,3-diketone groupδ C 196.8, δ C 117.5 and δ C 173.2, an oxygenated carbon delta C 91.5), a methine (δ C 41.9), a methylene group (δ C 28.2), two methyl groups (δ C 23.4 and δ C 28.1) The remaining 22 carbons can be assigned to benzoyl, one isoprenyl and one geranyl. Figure 4 ) shows that C-4 / C-5 / C-6 are connected and C-2 / C-3 / C-4 are connected. Combined with the 1,3-diketone unit C-6 / C-1 / C-2, it can be determined that compound I has a phloroglucinol core skeleton. 2 -14(δ H 1.70 and 1.62) and C-2 (δ C 173.2),C-3(δ C 42.5),C-4(δ C 207.3) and H-15 (δ H 2.64) and C-4 (δ C 207.3) showed that compound I had a cyclopropane structure. 2 -19 and H 2 The HMBC correlation of -24 with C-4, C-5 and C-6 proved that the isopentenyl and geranyl groups were connected through C-5, and the benzoyl group was connected to C-1. H The olefin proton signals at 5.58 (1H, m, H-29) and 5.58 (1H, m, H-30) and δ C The oxygen-containing carbon signal at 70.6 (C-31), as well as the HMBC correlation signals of H-30 with C-29, C-31, C-32 and C-33; H-29 with C-30 and C-31; and H-28 with C-29 determined that compound I had a hydroxyl group at C-31. C 173.2) and C-16 (δ C 91.5) and unsaturation analysis, it is inferred that the compound has a five-membered ether ring. It can be seen that compound I is a polycyclic polyisopentenyl phloroglucinol derivative with a furan ring-fused spiro[2.5]octane core, named Hypehenol B. The compound structure is shown in Figure 5 The structural formula of the compound with atomic numbers is shown in Figure 6 .

[0041] Table 1 Compound Ⅰ 1 H NMR and 13 C NMR data ( 1 H NMR 500 MHz, 13 C NMR 125MHz, in CDCl 3 , δinppm, Jin Hz)

[0042]

[0043] Example 3 Determination of anti-inflammatory activity of compound I

[0044] (1) Anti-inflammatory activity assay The RAW264.7 cell model was stimulated with LPS. RAW264.7 cells were cultured in high-glucose DMEM medium (Shanghai Yuanpei Biotechnology Co., Ltd.) containing 10% fetal bovine serum (Vicente Biotechnology (Nanjing) Co., Ltd.) at 37°C and 5% CO 2 When the cell density reaches 70%-80%, the subsequent experiments are carried out.

[0045] (2) 300 μL of RAW264.7 cells (6×10 4 The culture medium (100 μM / well) was inoculated into a 48-well cell culture plate. After overnight, the culture medium was aspirated and replaced with 300 μL of culture medium (high-glucose DMEM medium containing 10% fetal bovine serum) containing a series of concentrations of compound I (25, 12.5, 6.25, 3.125 μM) or indomethacin (50, 25, 12.5, 6.25 μM), and the cells were cultured for another hour, and then 30 μL of LPS (1 μg / mL) was added for co-culture for 24 hours.

[0046] (3) Take 50 μL of cell supernatant from each well and transfer it to a 96-well plate. Then, according to the instructions of the nitric oxide detection kit (Shanghai Biotech Biotechnology Co., Ltd.), first add 50 μL of room temperature Griess Reagent I, then add 50 μL of room temperature Griess Reagent II, incubate at 37°C for 5 min, and use an enzyme reader to measure the absorbance at a wavelength of 540 nm. According to the calculation formula, NO inhibition rate = (A540 LPS -A540 Sample ) / (A540 LPS -A540 Blank )]×100%, calculate the IC of NO inhibition rate 50 value.

[0047] A540 LPS: represents the absorbance value of the LPS stimulation group at a wavelength of 540nm. The LPS stimulation group is a group where only LPS is added to stimulate cells to produce a large amount of NO, serving as a positive control.

[0048] A540 Sample : represents the absorbance value of the sample group at a wavelength of 540nm. The sample group is the group to which the test compound and LPS are added, and is used to detect the inhibitory effect of the compound on NO generation.

[0049] A540 Blank : represents the absorbance value of the blank control group at a wavelength of 540nm. The blank control group is a group without LPS and test compounds, which is used to correct the background absorbance.

[0050] The results are shown in Table 2 and Figure 7 , IC of NO inhibition of compound Ⅰ 50 The value was 12.40±0.97μM, which was lower than the IC of the positive control drug Indomethacin. 50 The value was 16.08±0.98μM. Compound Ⅰ showed good anti-inflammatory activity.

[0051] Table 2 IC of compound Ⅰ and indomethacin in inhibiting LPS-induced NO production in RAW264.7 cells 50 value

[0052]

[0053] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polycyclic polyisopentenyl phloroglucinol compound having anti-inflammatory activity, characterized in that: The molecular structure is as follows:

2. The method for preparing a polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity according to claim 1, characterized in that: The specific steps are as follows: (1) The air-dried aerial part of Hypericum cyrtonema is made into powder, and the powder is extracted with dichloromethane at room temperature three times, each time for two days; the extract is concentrated and dried by a rotary evaporator to obtain a dichloromethane total extract; the mass volume ratio of the powder to the dichloromethane is 1:5; (2) The total dichloromethane extract obtained in step (1) was mixed with 200 g of 100-200 mesh silica gel, and 600 g of 200-300 mesh silica gel was used for column chromatography separation, and petroleum ether-ethyl acetate was used as the mobile phase for gradient elution, and the volume ratio of petroleum ether-ethyl acetate was 1:0, 50:1, 30:1, 15:1, 8:1, 4:1, 1.5:1, and 1:2; the eluate was collected and concentrated to dryness, and analyzed by thin layer chromatography, and similar components were combined to finally obtain 6 components Fr.A-Fr.F; (3) The Fr.D obtained in step (2) is subjected to a macroporous adsorption resin chromatography column and gradient eluted using 30%, 50%, 70% methanol aqueous solution and 100% methanol as mobile phases; the eluate is collected, analyzed by thin layer chromatography, and combined into Fr.D.1-Fr.D.6; (4) Fr.D.4 obtained in step (3) was eluted by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 10:1, 8:1, 4:1, 2:1, and 1:10 as the mobile phase, and the same components were combined to obtain Fr.D.4.1-Fr.D.4.5; Fr.D.4.2 was then eluted on a Sephadex LH-20 gel column using an eluent of dichloromethane:methanol = 1:1 to obtain five components Fr.D.4.2.1-Fr.D.4.2.5; (5) Fr.D.4.2.2 obtained in step (4) was purified by semi-preparative liquid chromatography using 70% methanol aqueous solution as the mobile phase to obtain compound I; the flow rate was 10 mL / min.

3. Use of the polycyclic polyisopentenyl phloroglucinol compound with anti-inflammatory activity as claimed in claim 1 in the preparation of anti-inflammatory drugs.

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