A 2,3-seco-guaianolide sesquiterpene lactone compound, a preparation method thereof, and uses thereof

Abstract

The present invention relates to a preparation method of 2,3-seco-guaianolide sesquiterpenoid compounds and their use in improving neuroinflammation. The 2,3-seco-guaianolide sesquiterpenoid compounds are extracted and extracted from the whole herb of Achillea millefolium L. using organic solvents, and then separated by two to three methods among normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and semi-preparative high-performance liquid chromatography to obtain a new monomeric 2,3-seco-guaianolide sesquiterpenoid compound. The monomeric compound is determined to be a new 2,3-seco-guaianolide sesquiterpenoid compound by methods such as high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, and its structure is identified. In addition, the activity of the 2,3-seco-guaianolide sesquiterpenoid compound in improving neuroinflammation is measured using microglial cell line BV2. The results show that the 2,3-seco-guaianolide sesquiterpenoid compound can improve neuroinflammation and can be used in anti-neuroinflammatory drugs. Achillea millefolium L.), and then separated by two to three methods among normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and semi-preparative high-performance liquid chromatography to obtain a new monomeric 2,3-seco-guaianolide sesquiterpenoid compound. The monomeric compound is determined to be a new 2,3-seco-guaianolide sesquiterpenoid compound by methods such as high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, and its structure is identified. In addition, the activity of the 2,3-seco-guaianolide sesquiterpenoid compound in improving neuroinflammation is measured using microglial cell line BV2. The results show that the 2,3-seco-guaianolide sesquiterpenoid compound can improve neuroinflammation and can be used in anti-neuroinflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a 2,3-seco-guaianolide compound, a preparation method thereof and uses thereof. Background Art

[0002] Microglia (BV2) are resident macrophage-like cells in the central nervous system and play a core role in the immune response of the central nervous system. Under the stimulation of injury or infection factors such as bacterial lipopolysaccharide (LPS), microglia are rapidly activated. Activated microglia can induce the activation of proteases, release various pro-inflammatory cytokines and glutamate, and generate a large amount of reactive oxygen species and reactive nitrogen species, resulting in damage and loss of surrounding other neurons and glial cells, causing and regulating neuroinflammatory responses, and ultimately may lead to neuronal death. The activation of microglia is involved in the occurrence and development of many neurodegenerative diseases. Therefore, many current studies are trying to play a neuroprotective role by inhibiting the release of inflammatory factors of BV2 microglia activated by LPS (bacterial lipopolysaccharide), and thus treating neurodegenerative diseases.

[0003] Achillea millefolium L. is a perennial herb of the genus Achillea in the family Asteraceae, and has a long medicinal history. It is widely used in traditional medicine to treat inflammation-related diseases such as pulmonary gastritis, nephritis, and skin inflammation. Sesquiterpene lactones, especially guaianolide-type sesquiterpene lactones, are one of the main secondary metabolites in Achillea millefolium L., and have a wide range of biological activities such as anti-inflammatory and anti-tumor activities. Although it has been found that guaianolide-type sesquiterpene lactones in Achillea millefolium L. have anti-inflammatory activities, there are no relevant reports on the improvement of neuroinflammation by guaianolide-type sesquiterpene lactones in Achillea millefolium L. at home and abroad. Summary of the Invention

[0004] The object of the present invention is to provide a 2,3-seco-guaianolide compound, a preparation method thereof and uses thereof. The 2,3-seco-guaianolide compound is obtained by extracting the whole herb of Achillea millefolium L. with an organic solvent extraction solvent, then performing extraction to obtain an extract, and then separating it by using two or three of normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and semi-preparative high performance liquid chromatography to obtain a new 2,3-seco-guaianolide monomer compound. Its structure is determined to be a new 2,3-seco-guaianolide compound by methods such as high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, and its structure is identified. The 2,3-seco-guaianolide compound described in the present invention is tested for its use in the preparation of a drug for improving neuroinflammation.

[0005] A 2,3-seco-guaianolide sesquiterpene lactone compound according to the present invention, the structural formula (I) of the compound is:

[0006]

[0007] Wherein: the compound of formula (I) is (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

[0008] The preparation method of the 2,3-seco-guaianolide sesquiterpene lactone compound is carried out according to the following steps:

[0009] a. Take the whole herb of Achillea millefolium as raw material, pulverize it, and carry out percolation, cold soaking extraction, soaking or heating reflux extraction at room temperature with 5-10 times the volume of ethanol aqueous solution with a volume concentration of 50-99%, methanol aqueous solution with a volume concentration of 50-99% or anhydrous methanol, and evaporate the solvent under reduced pressure to obtain the crude extract of Achillea millefolium;

[0010] b. Disperse the crude extract obtained in step a with water, and successively add petroleum ether, ethyl acetate and n-butanol in the system, or n-hexane, dichloromethane and n-butanol in the system, or petroleum ether, chloroform and n-butanol in the system, and extract 3-5 times. Concentrate the extraction solution of ethyl acetate, dichloromethane or chloroform to obtain an extract paste of ethyl acetate, dichloromethane or chloroform extract;

[0011] c. Separate the extract paste of ethyl acetate, dichloromethane or chloroform obtained in step b by two to three methods among normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography and semi-preparative high performance liquid chromatography, and then the compound of formula (I) ((2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone) can be obtained;

[0012] Among them, two separation methods:

[0013] The eluent for normal-phase silica gel column chromatography used is gradient elution with n-hexane-ethyl acetate or chloroform-methanol with a volume ratio of 100:1-0:1, and collect the fraction F with a flow ratio of 30:1-50:1. The fraction F is subjected to reverse-phase silica gel column chromatography or semi-preparative high performance liquid chromatography, and gradient elution is carried out with a methanol-aqueous solution with a concentration of 40%-99% to obtain the compound of formula I;

[0014] Three separation methods:

[0015] The eluent for the normal-phase silica gel column chromatography used is a gradient elution with a mixture of petroleum ether - acetone, dichloromethane - methanol, or chloroform - methanol with a volume ratio of 100:1 - 0:1. The fraction F with a ratio of 20:1 - 60:1 is collected. The fraction F is then subjected to reverse-phase silica gel column chromatography and gradient eluted with a methanol - aqueous solution with a concentration of 40% - 99% or an acetonitrile - aqueous solution with a concentration of 30% - 99%. The elution fraction F40 - 60 with 40 - 60% methanol - aqueous solution or acetonitrile - aqueous solution is collected. The fraction F40 - 60 is separated by semi-preparative high-performance liquid chromatography and isocratically eluted with a 30% acetonitrile - aqueous solution, an acetonitrile - aqueous solution with a concentration of 30% - 99%, or a 55% methanol - aqueous solution to obtain the compound of formula I.

[0016] The normal-phase silica gel column chromatography method used in step c is atmospheric pressure or pressurized column chromatography. The packing material used is normal-phase silica gel, and the eluent used is a mixture of at least two solvents selected from petroleum ether or n-hexane, acetone or ethyl acetate, chloroform or dichloromethane, and methanol, and isocratic elution or gradient elution is adopted.

[0017] The reverse-phase silica gel column chromatography method used in step c is atmospheric pressure or pressurized column chromatography. The eluent is a methanol aqueous solution with a volume concentration of 40 - 99% or an acetonitrile aqueous solution with a volume concentration of 30 - 99%, and isocratic elution or gradient elution is adopted.

[0018] The semi-preparative high-performance liquid chromatography method used in step c is pressurized column chromatography. The eluent is a methanol aqueous solution with a volume concentration of 40 - 99% or an acetonitrile aqueous solution with a volume concentration of 30 - 99%, and isocratic elution or gradient elution is adopted.

[0019] Use of the described 2,3-seco-guaiane-type sesquiterpene lactone compounds in the preparation of drugs for improving neuroinflammation.

[0020] A 2,3-seco-guaiane-type sesquiterpene lactone compound described in the present invention can be obtained by separation and purification from plants or synthesized by chemical modification methods well-known to those skilled in the art.

[0021] A 2,3-seco-guaiane-type sesquiterpene lactone compound described in the present invention is determined by modern spectroscopic means such as high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy. The structural identification process is as follows:

[0022] The compound of formula (I) is (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-oxo-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone, a colorless needle crystal, [α]20D 43.0 (c 0.1, MeOH); UV (MeOH) 218.1 nm; ECD (MeOH) 203 (Δε 31.97), 226 (Δε -40.84), 253 (Δε +8.95); its molecular formula C 19 H 24 O8 was determined by the quasi-molecular ion peak [M+Na]+ m / z 403.1358 (calcd. 403.1363) in its high-resolution mass spectrum; According to 1 H, 13 C NMR and two-dimensional nuclear magnetic resonance data, its structural skeleton type was determined to be 2,3-seco-guaiane sesquiterpene lactone compounds, named (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-oxo-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone; its 1 H and 13 C NMR assignments are shown in Table 1 [600 MHz ( 1 H), 150 MHz ( 13 C), CDCl3].

[0023] Table 1. 1 H and 13 C NMR data of the compound of formula (I) [δ (ppm), J (Hz)]

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the 1 H NMR (600 MHz, CDCl3) spectrum of the compound of formula (I) of the present invention;

[0026] Figure 2 is the 13 C NMR (150 MHz, CDCl3) spectrum of the compound of formula (I) of the present invention;

[0027] Figure 3 is the effect of the compound of formula (I) of the present invention on the viability of BV2 cells;

[0028] Figure 4Figure showing the effect of the compound of formula (I) of the present invention on the NO release in LPS-induced BV2 cells. Here, A represents the positive control drug andrographolide, and the data are expressed as mean ± standard deviation [****P<0.0001 vs. control group (C), ## P<0.01 vs. model group (LPS), #### P<0.0001 vs. model group (LPS)];

[0029] Figure 5 Figure showing the effect of the compound of formula (I) of the present invention on the inflammatory factor TNF-α in BV2 cells. The data are expressed as mean ± standard deviation [****P<0.0001 vs. control group (C), ## P<0.01 vs. model group (LPS), #### P<0.0001 vs. model group (LPS)];

[0030] Figure 6 Figure showing the effect of the compound of formula (I) of the present invention on the content of inflammatory factors in BV2 cells. Among them, Figure 6 A, 6B, and 6C are the contents of IL-18, PGE2, and IL-6 respectively; A represents the positive control drug andrographolide; the data are expressed as mean ± standard deviation [****P<0.0001 vs. control group (C), # P<0.05 vs. model group (LPS), ## P<0.01 vs. model group (LPS), #### P<0.0001 vs. model group (LPS)];

[0031] Figure 7 Figure showing the effect of the compound of formula (I) of the present invention on the protein expression level in BV2 cells. Among them, Figure 7 A and 7B are the effects of the compound of formula (I) on the protein expression levels of LOX, iNOS, and COX2 in BV2 cells respectively; Figure 7 C is the ratio of LOX to actin; 7D is the ratio of iNOS to actin; 7E is the ratio of COX2 to actin; A represents the positive control drug andrographolide. The data are expressed as mean ± standard deviation [**P<0.01 vs. control group (C), ****P<0.0001 vs. control group (C), # P<0.05 vs. model group (LPS), ## P<0.01 vs. model group (LPS), ### P<0.001 vs. model group (LPS), #### P<0.0001 vs. model group (LPS)];

[0032] Figure 8The effect of the compound of formula (I) of the present invention on the protein expression level in BV2 cells is shown in FIG. Figure 8 A is the effect of the compound of formula (I) on the expression levels of NLRP3, Caspase 1, IL-1β and ASC proteins in BV2 cells; Figure 8 B is the ratio of NLRP3 to GAPDH; Figure 8 C is the ratio of Caspase 1 to GAPDH; Figure 8 D is the ratio of IL-1β to actin; Figure 8 E is the ratio of ASC to GAPDH. A represents the positive control drug andrographolide; data are expressed as mean ± standard deviation [**P<0.01vs. control group (C), ****P<0.0001vs. control group (C), # P<0.05vs. model group (LPS), ## P<0.01vs. model group (LPS), ### P<0.001vs. model group (LPS), #### P<0.0001vs. model group (LPS)]. DETAILED DESCRIPTION

[0033] All reagents used were analytically pure, and acetonitrile in HPLC was HPLC grade (Merck, Germany). Normal phase silica gel for column chromatography (100-200 mesh, 200-300 mesh): produced by Qingdao Ocean Chemical Plant; reversed phase silica gel ODS: produced by Merck, Germany; HPLC (Dionex, USA) was configured as follows: P680HPLC pump, ASI-100 autosampler, TCC-100 column oven, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, and Chameleon chromatography workstation. The preparative high performance liquid chromatography (Dionex, USA) was configured as follows: P680HPLC pump, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, Chameleon chromatography workstation; mass spectra were measured using a quadrupole-time-of-flight hybrid mass spectrometer (Applied Biosystems, USA); nuclear magnetic resonance was measured using a VARIAN VNMRS 600 MHz nuclear magnetic resonance spectrometer; and ECD spectra were obtained using a Chirascan spectropolarimeter (UK).

[0034] The whole herb of Achillea millefolium was collected from Changji area of ​​Xinjiang Uygur Autonomous Region and was identified as Achillea millefolium L. by Associate Researcher Lu Chunfang from Xinjiang Technical Institute of Physics and Chemistry of the Chinese Academy of Sciences.

[0035] Example 1

[0036] a. Take 5 kg of the whole plant raw material of Achillea millefolium, crush it, and perform cold extraction at room temperature with 5 times the volume of an ethanol aqueous solution with a concentration of 50%. Evaporate the solvent under reduced pressure to obtain the crude extract of Achillea millefolium;

[0037] b. Disperse the crude extract obtained in step a with water, and sequentially add petroleum ether, ethyl acetate, and n-butanol for extraction 3 times. Combine the ethyl acetate layer and evaporate it to dryness under reduced pressure to obtain the ethyl acetate layer extract paste;

[0038] c. Gradient elute the ethyl acetate extract paste obtained in step b with a petroleum ether-acetone eluent with a ratio of 100:1 - 0:1 on a normal-phase silica gel column, and collect fraction F with a ratio of 20:1; Fraction F is subjected to reverse-phase silica gel column chromatography and gradient eluted with a methanol-aqueous solution with a concentration of 40% - 99%, and collect the 50% methanol-aqueous solution elution fraction F50; Fraction F50 is subjected to semi-preparative high-performance liquid chromatography and isocratically eluted with a 30% acetonitrile-aqueous solution to obtain the compound of formula I (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

[0039] Example 2

[0040] a. Take 5 kg of the whole plant raw material of Achillea millefolium, crush it, and perform reflux extraction at 80 °C with 10 times the volume of an ethanol aqueous solution with a concentration of 99%. Evaporate the solvent under reduced pressure to obtain the crude extract of Achillea millefolium;

[0041] b. Disperse the crude extract obtained in step a with water, and sequentially add n-hexane, dichloromethane, and n-butanol for extraction 4 times. Combine the dichloromethane layer and evaporate it to dryness under reduced pressure to obtain the dichloromethane extract paste;

[0042] c. Gradient elute the dichloromethane extract paste obtained in step b with a n-hexane-ethyl acetate eluent with a volume ratio of 100:1 - 0:1 on a normal-phase silica gel column, and collect fraction F with a ratio of 30:1; Fraction F is subjected to reverse-phase silica gel column chromatography and gradient eluted with a methanol-aqueous solution with a concentration of 40% - 99% to obtain the compound of formula I (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

[0043] Example 3

[0044] a. Take 5 kg of the whole plant raw material of Achillea millefolium, crush it, and perform percolation extraction at room temperature with 6 times the volume of a methanol aqueous solution with a concentration of 99%. Evaporate the solvent under reduced pressure to obtain the crude extract of Achillea millefolium;

[0045] b. Disperse the crude extract obtained in step a with water, successively add petroleum ether, chloroform, and n-butanol for extraction 5 times, combine the chloroform layer and evaporate it to dryness under reduced pressure to obtain the chloroform extract extract;

[0046] c. Use normal-phase silica gel column chromatography to elute the chloroform extract extract obtained in step b with a gradient of chloroform-methanol with a volume ratio of 100:1 - 0:1, collect fraction F with a ratio of 50:1. Fraction F is subjected to semi-preparative high-performance liquid chromatography and eluted with a gradient of methanol-aqueous solution with a concentration of 40% - 99% to obtain the compound of formula I (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

[0047] Example 4

[0048] a. Take 5 kg of the raw material of Achillea millefolium whole herb, crush it, and perform percolation extraction at room temperature with 7 times the volume of a 50% methanol aqueous solution. Evaporate the solvent to dryness under reduced pressure to obtain the crude extract of Achillea millefolium;

[0049] b. Disperse the crude extract obtained in step a with water, successively add n-hexane, dichloromethane, and n-butanol for extraction 5 times, combine the dichloromethane layer and evaporate it to dryness under reduced pressure to obtain the dichloromethane extract extract;

[0050] c. Use normal-phase silica gel column chromatography to elute the dichloromethane extract extract obtained in step b with a gradient of dichloromethane-methanol with a volume ratio of 100:1 - 0:1, collect fraction F with a ratio of 60:1. Fraction F is subjected to reverse-phase silica gel column chromatography and eluted with a gradient of acetonitrile-aqueous solution with a concentration of 30% - 99%. Collect the elution fraction F40 with 40% acetonitrile-aqueous solution. Fraction F40 is subjected to semi-preparative high-performance liquid chromatography and eluted with a gradient of acetonitrile-aqueous solution with a concentration of 30% - 99% to obtain the compound of formula I (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

[0051] Example 5

[0052] a. Take 5 kg of the raw material of Achillea millefolium whole herb, crush it, and soak and extract it with 8 times the volume of anhydrous methanol at room temperature. Evaporate the solvent to dryness under reduced pressure to obtain the crude extract of Achillea millefolium;

[0053] b. Disperse the crude extract obtained in step a with water, successively add petroleum ether, ethyl acetate, and n-butanol for extraction 3 times, combine the ethyl acetate layer and evaporate it to dryness under reduced pressure to obtain the ethyl acetate extract extract;

[0054] c. The ethyl acetate extract obtained in step b was eluted by gradient elution with chloroform-methanol having a volume ratio of 100:1 - 0:1 on a normal-phase silica gel column, and fraction F with a ratio of 50:1 was collected. Fraction F was subjected to reverse-phase silica gel column chromatography and eluted with a gradient of methanol-aqueous solution with a concentration of 40% - 99%, and the elution fraction F60 with 60% acetonitrile-aqueous solution was collected. Fraction F60 was subjected to semi-preparative high-performance liquid chromatography and isocratically eluted with a methanol-aqueous solution with a concentration of 55% to obtain the compound of formula I (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

[0055] Example 6

[0056] Application of the 2,3-seco-guaiane sesquiterpene lactone compound isolated from the whole herb of Achillea millefolium in the preparation of a drug for improving neuroinflammation, taking microglia (BV2) as an example;

[0057] Cell culture:

[0058] BV2 cells (purchased from BeNa Culture Collection, BNCC) were cultured in Dulbecco's modified eagle medium (DMEM) high-glucose medium containing 10% fetal bovine serum (FBS) (purchased from Gibco, USA), 1% penicillin and streptomycin in an incubator at 37°C and 5% CO2;

[0059] Test on the effect of the compound of the present invention on cell viability:

[0060] The compound of formula I was dissolved in dimethyl sulfoxide (DMSO). BV2 cells in the logarithmic growth phase with good growth state were seeded into a 96-well plate at 5×10 3 cells / well. In the experimental group, different concentrations of 12.5, 25, 50 and 100 μM of the compound of formula I were added, and in the control group, dimethyl sulfoxide (DMSO) was added. After culturing for 24 hours, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (CCK-8 reagent) was added to each well, and the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell survival rate;

[0061] Determination of nitric oxide (NO) content:

[0062] The Griess method was used to test the release of NO in BV2 cells. Samples of different concentrations of 25, 50 and 100 μM were added and incubated for 2 hours, and then 1 μg / mL lipopolysaccharide (LPS) was added and incubated for 22 hours. After the incubation was completed, the cell supernatant was collected and the content of nitric oxide in the cell supernatant was determined by the Griess method. Before the determination, Griess Reagent I and II were taken out and restored to room temperature. The standard (1-100 μM) was diluted with complete culture medium. The concentration of the standard can be 0, 1, 2, 5, 10, 20, 40, 60, 100 μM. The standard and the collected culture supernatant were added to a 96-well plate at 50 μL / well. 50 μL of Griess Reagent I restored to room temperature and 50 μL of Griess Reagent were added to each well in turn. II. After 5 minutes of shaking and mixing, the absorbance was measured at 540 nm, and a standard curve was made. The NO content in the culture supernatant was calculated based on the standard curve;

[0063] Enzyme-linked immunosorbent assay (ELISA) for detection of prostaglandin E2 (PGE2), interleukin-18 (IL-18), and interleukin-6 (IL-6)

[0064] and inflammatory factors such as tumor necrosis factor-α (TNF-α):

[0065] BV2 cells were incubated with drugs at different concentrations of 25, 50 and 100 μM for 2 hours, and then 1 μg / mL of lipopolysaccharide (LPS) was added for 22 hours. The cell culture medium was collected and the contents of PGE2, IL-18, IL-6 and TNF-α were tested according to the instructions of the corresponding ELISA kits.

[0066] Western blotting was used to detect the expression levels of lipoxygenase (LOX), cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS), nucleotide oligomerization domain (NOD)-like receptor pyrin domain-related protein 3 (NLRP3) and other proteins:

[0067] BV2 cells were placed in a 6-well plate overnight, then different concentrations of drugs (25, 50, and 100 μM) were added and incubated for 2 hours. Then, 1 μg / mL of LPS was added and co-incubated for 22 hours. BV2 cells were collected using lysis buffer (Radio-Immunoprecipitation Assay Buffer, RIPA), and the protein concentration was detected using a protein quantification kit (Bicinchoninic acid, BCA). All samples containing equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins in the gel were transferred to a polyvinylidenefluoride (PVDF) membrane. The membrane was incubated in a TBST (Tris-buffered saline with Tween) buffer containing 5% milk for 1 hour to prevent non-specific protein binding. The primary antibody was added and incubated overnight on a shaker at 4°C. The membrane was washed 3 times with TBST buffer, then incubated with a horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour at room temperature. The membrane was washed 3 times with TBST buffer, 10 minutes each time. An enhanced chemiluminescence reagent was added for development. Nordihydroguaiaretic acid (NDGA) is a positive control drug for the lipoxygenase protein LOX, and andrographolide is a positive control drug for other proteins.

[0068] Experimental results:

[0069] As Figure 3 shown, the compound of formula (I) had no obvious cytotoxicity to BV2 cells at the tested concentrations; the compound of formula (I) could significantly inhibit the NO release in LPS-induced BV2 cells at four tested concentrations (12.5, 25, 50, and 100 μM) (P < 0.0001), and showed concentration dependence ( Figure 4 ); the compound of formula (I) significantly reduced the levels of TNF-α (P < 0.0001) ( Figure 5 ), IL-18, PGE2, and IL-6 ( Figure 6 ) in BV2 cells; in addition, the compound of formula (I) could inhibit the protein expression levels of LOX, iNOS, and COX2 in LPS-induced BV2 cells ( Figure 7 ), could significantly inhibit the protein expression levels of ASC and IL-1β in a concentration-dependent manner, could inhibit the protein expression level of NLRP3 at 100 μM, and had no inhibitory activity on Caspase 1 at all tested concentrations.

[0070] The above results indicate that the compound of formula (I) can reduce the release of inflammatory factors in LPS-induced BV2 microglia, significantly inhibit the expression of related proteins, alleviate neuroinflammatory responses, has the effect of improving neuroinflammation, and can be used for the pharmaceutical use of treating neuroinflammation-related diseases.

Claims

1. A 2,3-seco-guaiane sesquiterpene lactone compound, characterized in that The structure of the compound is shown in formula (I): Wherein: the compound of formula (I) is (2S,6S,7R,8S,10S,11S)-2-hydroxy-4-one-10-hydroxy-15-oxo-8-[(E)-2-methyl-2-butenoyloxy]-guaia-1-ene-12,6-lactone.

2. Use of the 2,3-seco-guaiane sesquiterpene lactone compound according to claim 1 in the preparation of a drug for improving neuroinflammation.

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