Sesquiterpene compound as well as preparation method and application thereof

Abstract

The invention discloses a sesquiterpene compound as well as a preparation method and application thereof, and relates to the technical field of medicines. The invention provides a sesquiterpene compound and discloses new activity of the sesquiterpene compound, namely the sesquiterpene compound is used for remarkably inhibiting generation of inflammatory BV2 cell NO and remarkably inhibiting oxidative stress SH-SY5Y cell damage. The sesquiterpene compound can significantly inhibit TLR4 / NF-kappa B signal channels to inhibit expression of downstream inflammatory protein nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and has an application prospect in treatment of neuroinflammatory diseases. The invention can obtain the sesquiterpene compound as well as the preparation method and the application thereof.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a sesquiterpene compound, a preparation method thereof, and an application thereof. Background Art

[0002] Neuroinflammation is a complex neural cascade reaction by which the biological body defends against infections and prevents internal injuries. It can induce or even exacerbate various neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.). Therefore, neuroinflammation is considered an important factor in neuronal degenerative diseases. [1] It shows that inhibiting the neuroinflammatory response can help reduce neuronal lesions in the damaged brain regions. [2] Parkinson's disease (PD) is the second most common neurodegenerative disease in humans globally. Currently, there is still no effective cure, and only drugs can be used to delay its progression. [3] Factors such as the aging population, lack of treatment methods, and difficulty in targeted drug use have made the treatment of such elderly chronic diseases more severe. [4] Therefore, deeply revealing the pathogenesis of PD and finding effective therapeutic targets will provide new strategies for the treatment of PD. Apoptosis is a process of cell autonomous and orderly death regulated by genes. The Bcl-2 family [5] (B-cell lymphoma / leukemia-2-like protein) is an important apoptosis regulatory protein that regulates this process. It can be divided into two categories: anti-apoptotic genes such as the Bcl-2 gene and pro-apoptotic genes such as the Bax gene. Caspase-3 is a key protease in the process of apoptosis. Its mechanism is to regulate the Bcl2 / Bax / Caspase3 signaling pathway, down-regulate the expression of Bax and Caspase-3, and at the same time increase the expression level of Bcl-2 to antagonize the oxidative damage caused.

[0003] Cyclooxygenase-2 (COX-2) is the key enzyme for the synthesis of prostaglandin E2. Increased COX-2 immunoreactivity has been found in pyramidal cells of the cerebral cortex and the hippocampus, suggesting that COX-2 may be involved in the pathogenesis of Alzheimer's disease. And COX-2 is mainly expressed in inflammatory sites and can cause apoptosis of nerve cells. The increased expression of COX-2 in the brains of AD patients is highly consistent with the level of Aβ, and COX-2 coexists with neurons containing NFTs, indicating that the increased expression of COX-2 in neurons may lead to neuronal death.

[0004] Therefore, in order to develop new anti-neuroinflammatory drugs for the treatment of Alzheimer's disease, it is urgent to find COX-2 enzyme inhibitors. [6]

[0005] ​[1] Zhang Rong, Su Zhaoliang, Zhao Wenjuan, et al. Research progress on natural products against neuroinflammation [J]. Chinese Traditional and Herbal Drugs, 2014, 45(5): 730-737.

[0006] [2] Qiu Aowang, Liu Zhan, Guo Jun, et al. Relationship between neuroinflammation and neurodegenerative diseases [J]. Progress in Physiological Sciences, 2011, 42(5): 353-358.

[0007] [3] Weintraub D, Aarsland D, Chaudhurik R, et al. The neuropsychiatry of Parkinson's disease: Advances and challenges [J]. Lancet Neurol, 2022, 21(1): 89-102.

[0008] [4] Zhang Shuo, Gao Jian, Jiang Ligang. Current status of epidemiological research on the prevalence and related factors of Parkinson's disease [J]. Journal of Jilin Medical College, 2021, 42(6): 437-439.

[0009] [5] Chen Y, Song T, Sun X, et al. Regulatory effect of Angelica sinensis polysaccharide on Bcl-2 / Bax / Caspase-3 signal pathway in spleen of rats with radiation injury [J]. Journal of Biobased Materials and Bioenergy, 2020, 14(4): 579-583.

[0010] [6] Jiang Zhenzhou, Xu Dengqiu, Yang Hang, et al. Research progress on drug action targets [J]. Progress in Pharmaceutical Sciences, 2018, 42(11): 838-859. Summary of the Invention

[0011] The object of the present invention is to solve the above technical problems, and to provide a sesquiterpene compound, a preparation method and an application thereof.

[0012] A sesquiterpene compound, the chemical formula of the sesquiterpene compound is C 15 H 22 O4, and the structural formula of the sesquiterpene compound is as follows:

[0013]

[0014] A preparation method of a sesquiterpene compound is carried out according to the following steps:

[0015] Step 1: Inoculate Hericium erinaceus into a PDA plate medium at an inoculation amount of 5%; activate and culture it at 28°C for 7 days to obtain an activated bacterial cake;

[0016] Step 2: Inoculate the activated bacterial cake obtained in Step 1 onto a rice medium at an inoculation amount of 5%; culture it at 28°C for 32 days to obtain a rice fermentation medium;

[0017] Step 3: Extract the rice fermentation medium obtained in Step 2 with methanol to obtain a crude extract; extract the crude extract 3 times with a water-ethyl acetate mixed solution to obtain an extracted crude extract; fractionate with a silica gel column and sequentially use chloroform-methanol mixed solutions with a volume ratio of chloroform to methanol of (100:1), (50:1), (25:1), (10:1), (5:1) and MeOH solution for gradient elution to obtain fraction a containing the target product; separate fraction a containing the target product through a chromatographic column to obtain fraction b containing the target product; purify fraction b containing the target product through a gel column, a silica gel column and a semi-preparative high performance liquid chromatograph in sequence to obtain a sesquiterpene compound.

[0018] An application of a sesquiterpene compound, the application of the sesquiterpene compound in the preparation of an anti-neuroinflammatory drug, a drug for inhibiting the production of NO by inflammatory BV2 microglia, a drug for inhibiting downstream inflammatory proteins by inhibiting the TLR4 / NF-κB signaling pathway, a drug for inhibiting the apoptotic activity of SH-SY5Y cells, and a drug for promoting Bcl-2 expression and inhibiting Bax protein expression, wherein the neuroinflammation includes but is not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease or Parkinson's disease.

[0019] Advantages of the present invention:

[0020] The present invention provides a sesquiterpene compound and discloses its new activity, namely, it is used to significantly inhibit the production of NO in inflammatory BV2 cells and significantly inhibit the damage of oxidative stress SH-SY5Y cells. This sesquiterpene compound significantly inhibits the TLR4 / NF-κB signaling pathway to inhibit the expression of downstream inflammatory proteins nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), with inhibition rates of 86.4% and 85.0% respectively; and the inhibition rate of inhibiting NO production in inflammatory BV2 cells is 56%; this sesquiterpene compound significantly inhibits the expression of phosphorylated NF-κB p65 protein stimulated by LPS, with an inhibition rate of 90.0%; at the same time, this sesquiterpene compound can significantly inhibit the damage of oxidative stress SH-SY5Y cells, and its cell survival rate is 91.6%; after adding this sesquiterpene compound, the up-regulation rate of Bcl-2 protein expression is 34.6%, and the down-regulation rate of Bax protein expression is 27.6%, effectively preventing cell apoptosis. In summary, it shows that this sesquiterpene compound has the application prospect of treating neuroinflammatory diseases.

[0021] The present invention can obtain a sesquiterpene compound, its preparation method and application. Brief Description of the Drawings

[0022] Figure 1 It is the hydrogen spectrum diagram of the sesquiterpene compound of the present invention;

[0023] Figure 2 It is the carbon spectrum diagram of the sesquiterpene compound of the present invention;

[0024] Figure 3 It is the ESI-MS diagram of the sesquiterpene compound of the present invention;

[0025] Figure 4 The influence diagram of the sesquiterpene compound of the present invention on the production of NO induced by LPS in BV-2;

[0026] Figure 5 The influence diagram of the sesquiterpene compound of the present invention on the protein level of enzyme markers in LPS-stimulated BV-2 cells;

[0027] Figure 6 The influence diagram of the sesquiterpene compound of the present invention on inhibiting the damage activity of H2O2-induced SH-SY5Y cells;

[0028] Figure 7 The influence diagram of the sesquiterpene compound of the present invention on the protein level of enzyme markers in H2O2-induced SH-SY5Y cells. Detailed Embodiments

[0029] Detailed Embodiment 1: A sesquiterpene compound in this embodiment, the chemical formula of the sesquiterpene compound is C 15 H 22 O4, and the structural formula of the sesquiterpene compound is as follows:

[0030]

[0031] Specific Embodiment 2: A method for preparing a sesquiterpene compound according to this embodiment is carried out according to the following steps:

[0032] Step 1: Inoculate Hericium erinaceus on a PDA plate medium with an inoculation amount of 5%; activate and culture at 28°C for 7 days to obtain an activated cake of bacteria;

[0033] Step 2: Inoculate the activated cake of bacteria in Step 1 onto a rice medium with an inoculation amount of 5%; culture at 28°C for 32 days to obtain a rice fermentation medium;

[0034] Step 3: Extract the rice fermentation medium in Step 2 with methanol to obtain a crude extract; extract the crude extract 3 times with a water-ethyl acetate mixed solution to obtain an extracted crude extract; segment with a silica gel column, and sequentially use chloroform-methanol mixed solutions with a volume ratio of chloroform to methanol of (100:1), (50:1), (25:1), (10:1), (5:1) and MeOH solution for gradient elution to obtain fraction a containing the target product; separate fraction a containing the target product through a chromatographic column to obtain fraction b containing the target product; purify fraction b containing the target product through a gel column, a silica gel column and a semi-preparative high performance liquid chromatograph in sequence to obtain the sesquiterpene compound.

[0035] Advantages of this embodiment:

[0036] This embodiment provides a sesquiterpene compound and discloses its new activity, that is, it is used to significantly inhibit the production of NO in inflammatory BV2 cells and significantly inhibit the damage of oxidative stress SH-SY5Y cells. This sesquiterpene compound significantly inhibits the TLR4 / NF-κB signaling pathway to inhibit the expression of downstream inflammatory proteins nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the inhibition rates are 86.4% and 85.0% respectively; and the inhibition rate of inhibiting NO production in inflammatory BV2 cells is 56%; this sesquiterpene compound significantly inhibits the expression of phosphorylated NF-κB p65 protein stimulated by LPS, and the inhibition rate is 90.0%; at the same time, this sesquiterpene compound can significantly inhibit the damage of oxidative stress SH-SY5Y cells, and its cell survival rate is 91.6%; after adding this sesquiterpene compound, the up-regulation rate of Bcl-2 protein expression is 34.6%, and the down-regulation rate of Bax protein expression is 27.6%, effectively preventing cell apoptosis. In summary, it shows that this sesquiterpene compound has the application prospect of treating neuroinflammatory diseases.

[0037] Specific Embodiment 3: The difference between this embodiment and Specific Embodiment 1 or 2 is that the Hericium erinaceus used in Step 1 was purchased from the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the number 5.579.

[0038] Other steps are the same as those in Specific Embodiment 1 or 2.

[0039] Specific Embodiment 4: The difference between this embodiment and any one of Specific Embodiments 1 to 3 is that the 1000 mL PDA plate medium in Step 1 contains 10 g of peptone, 40 g of glucose, and 20 g of agar powder.

[0040] Other steps are the same as those in Specific Embodiments 1 to 3.

[0041] Specific Embodiment 5: The difference between this embodiment and any one of Specific Embodiments 1 to 4 is that the rice medium described in Step 2 contains 100 g of rice and 60 mL of sterile water.

[0042] Other steps are the same as those in Specific Embodiments 1 to 4.

[0043] Specific Embodiment 6: The difference between this embodiment and any one of Specific Embodiments 1 to 5 is that in Step 3, the water-ethyl acetate mixed solution has water and ethyl acetate mixed in equal volumes.

[0044] Other steps are the same as those in Specific Embodiments 1 to 5.

[0045] Specific Embodiment 7: The difference between this embodiment and any one of Specific Embodiments 1 to 6 is that the volume ratios of the chloroform-methanol mixed solution and the MeOH solution for gradient elution in Step 3 are 200:200:200:100:100:100 in sequence.

[0046] Other steps are the same as those in Specific Embodiments 1 to 6.

[0047] Specific Embodiment 8: The difference between this embodiment and any one of Specific Embodiments 1 to 7 is that the solvent in the chromatographic column in Step 3 is a methanol-water mixed solution, and the elution gradient of methanol is 10% - 100%.

[0048] Other steps are the same as those in Specific Embodiments 1 to 7.

[0049] Specific Embodiment 9: The difference between this embodiment and any one of Specific Embodiments 1 to 8 is that the solvent in the gel column in Step 3 is methanol; the solvent in the silica gel column is a petroleum ether-ethyl acetate solution, and the volume ratio of petroleum ether to ethyl acetate is 9:1; the solvent in the semi-preparative high performance liquid chromatograph is a methanol-water mixed solution, the mass fraction of methanol is 40%, and the flow rate of the high performance liquid column is 2 mL / min.

[0050] The other steps are the same as those in the first to eighth specific embodiments.

[0051] Specific Embodiment Ten: The application of a sesquiterpene compound. The sesquiterpene compound is used in the preparation of drugs for anti-neuroinflammation, drugs for inhibiting the production of NO by inflammatory BV2 microglial cells, drugs for inhibiting downstream inflammatory proteins by inhibiting the TLR4 / NF-κB signaling pathway, drugs for inhibiting the apoptotic activity of SH-SY5Y cells, and drugs for promoting the expression of Bcl-2 and inhibiting the expression of Bax protein. The neuroinflammation includes but is not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, or Parkinson's disease.

[0052] The following examples are used to verify the beneficial effects of the present invention:

[0053] Example 1:

[0054] I. Extraction method, identification, anti-neuroinflammation determination and application of the compound in this example:

[0055] 1. Experimental materials:

[0056] Reagents and instruments:

[0057] Common organic solvents: chloroform, dichloromethane, methanol, ethyl acetate, petroleum ether, acetone, etc. are all industrial reagents. Organic solvents: dimethyl sulfoxide (DMSO), chromatographic methanol, chromatographic acetonitrile, etc.

[0058] Common instruments: Rudolph Autopol Ⅲ polarimeter; High performance liquid chromatograph: LC-20AT; Ultraviolet spectrometer: Thermo Evolution-300; Infrared spectrometer: Bruker TENSOR 27 (potassium bromide tablet method); Nuclear magnetic resonance: Bruker Avance Ⅲ 500 (TMS internal standard); Low resolution mass spectrometer: Thermo Fisher LTQ Fleet; High resolution mass spectrometer: Agilent 6520 Accurate-Mass Q-TOF LC / MS. Rotary evaporator: RE-2000A; Low temperature coolant circulating pump: DLSB-10 / 20 (Zhengzhou Great Wall Scientific Industry and Trade Co., Ltd.); Circulating water type multi-purpose vacuum pump: SHB-Ⅲ (Zhengzhou Great Wall Scientific Industry and Trade Co., Ltd.); Ultra-clean workbench: SW-OJ-2F (Suzhou Antai Air Technology Co., Ltd., Sujing Group); Vertical steam sterilizer: MLS-3780. Column chromatography silica gel (100 - 200 mesh, 200 - 300 mesh and 300 - 400 mesh) and thin layer chromatography silica gel (silica gel H) are all produced by Qingdao Marine Chemical Factory; Liquid chromatography column: Hypersil BDS 5μm C18 (250×4.6 and 250×10; Thermo); Hydroxypropyl dextran gel Sephadex LH-20 and RP-C18 reverse silica gel are all produced by Merk.

[0059] 1.1 Fermentation culture of bacteria:

[0060] Inoculate Hericium erinaceus in PDA plate medium (potato dextrose agar) with an inoculation amount of 5%; Activate and culture at 28°C for 7 days until the petri dish is covered with mycelium to obtain an activated bacterial cake;

[0061] Hericium erinaceus was purchased from the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the number 5.579, and the purchase website https: / / cgmcc.net / directory / data?number=5.579&genus=&species=&yiming =&page=1 .

[0062] 1000 mL of PDA plate medium contains 10 g of peptone, 40 g of glucose and 20 g of agar powder.

[0063] Inoculate the activated bacterial cake (7 mm 2 ) on the rice medium in a 500 mL fermentation flask with an inoculation amount of 5%; Culture at 28°C for 32 days to obtain a rice fermentation medium;

[0064] The rice medium contains 100 g of rice and 60 mL of sterile water, and a total of 10 kg of rice is fermented.

[0065] 1.2 Compound extraction and separation:

[0066] The rice fermentation medium was extracted with methanol, and the methanol was evaporated to obtain the crude extract; the crude extract was extracted 3 times with 5 L of a water-ethyl acetate mixed solution to obtain 19.0 g of the extracted crude extract; it was fractionated with a silica gel column, and chloroform-methanol mixed solutions with volume ratios of chloroform to methanol of (100:1), (50:1), (25:1), (10:1), (5:1) and MeOH solution were used for gradient elution in sequence to obtain 6 fractions F1 - F6; fraction F4 was separated by an RP-18 chromatographic column to obtain 5 fractions F4-1 to F4-5; fraction F4-2 was purified successively through an LH-20 gel column, a silica gel column and a semi-preparative high performance liquid chromatograph (HPLC) to obtain a sesquiterpene compound (t R = 11 min, 8.0 mg), named: 3β,6α-dihydroxycinnamolide.

[0067] Note: Both the above fraction F4 and fraction F4-2 contain the target sesquiterpene compound, which was determined by detection with a nuclear magnetic resonance instrument and a mass spectrometer.

[0068] In the water-ethyl acetate mixed solution, water and ethyl acetate were mixed in equal volumes;

[0069] The volume ratios of the chloroform-methanol mixed solution and MeOH solution for gradient elution were 200:200:200:100:100:100 in sequence;

[0070] The solvent in the chromatographic column was a methanol-water mixed solution, and the elution gradient of methanol was 10% - 100%;

[0071] The solvent in the gel column was methanol; the solvent in the silica gel column was a petroleum ether-ethyl acetate solution, and the volume ratio of petroleum ether to ethyl acetate was 9:1; the solvent in the semi-preparative high performance liquid chromatograph was a methanol-water mixed solution, the mass fraction of methanol was 40%, and the flow rate of the high performance liquid column was 2 mL / min.

[0072] 1.3 The physical and chemical properties of the sesquiterpene compound are as follows:

[0073] The physical and chemical properties of the sesquiterpene compound obtained in this example are: the molecular formula is C 15 H 22 O4, yellow oily, and the structural formula is as follows:

[0074]

[0075] ESI-MS(positive) m / z: 267.00 [M+H] + .

[0076] 1H-NMR(500MHz, CD3OD) δ: 6.66(1H, m, H-7), 4.57(1H, m, H-6), 4.46(1H, t, J = 9.3Hz, H-11a), 4.12(1H, t, J = 8.9Hz, H-11b), 3.27(1H, m, H-3), 2.98(1H, m, H-8), 1.69(1H, m, H-2a), 1.67(1H, m, H-5), 1.52(1H, m, H-1a), 1.48(1H, m, H-2b), 1.46(1H, m, H-1b), 1.28(1H, s, H-14), 1.04(1H, s, H-15), 0.89(1H, s, H-13);

[0077] 13 C-NMR(125MHz, CD3OD) δ: 172.2(C-12), 137.9(C-7), 129.6(C-8), 79.4(C-3), 68.8(C-11), 68.6(C-6), 57.7(C-5), 50.9(C-9), 41.0(C-10), 40.4(C-4), 37.9(C-1), 30.9(C-14), 27.7(C-2), 16.3(C-15), 14.7(C-13).

[0078] 1.4 Nitric oxide (Lactate dehydrogenase, NO) inhibitory activity:

[0079] NO is an important inflammatory mediator. In neurons and glial cells of the brains of patients with neurodegenerative diseases, the overproduction of NO is common, indicating that NO is involved in such neuroinflammation [7] . Evaluate the inhibitory effect of the compound on the production of NO by LPS-induced BV-2 microglia. Before adding the detection reagent, BV-2 cells (2×10 5(Cells at a density of 1×10⁵ cells / mL) were seeded in a 96-well plate and cultured at 37 °C for 24 hours. The experimental groups were divided into: a control group (DMSO), an LPS treatment group (1 μg / mL LPS), and a compound treatment group (1 μg / mL LPS + 50 μM compound). After adding the drugs, the cells were incubated for another 24 hours. The concentration of nitrite in the culture medium was measured according to the protocol using a commercial assay kit. The main principle was to detect the release of NO in the culture medium by the Griess method to determine the anti-neuritis activity of the compound. The supernatant (50 μL) of BV-2 cells was reacted with Griess reagent (50 μL Griess regent I and 50 μL Griess regent II). The absorbance was measured at 540 nm on a Bio-Tek microplate reader, and the nitrite concentration was calculated through a nitrite standard curve using sodium nitrite as the standard.

[0080] Inhibition rate % = (A LPS组 - A 实验组 ) / (A LPS组 - A 空白组 ) × 100;

[0081] Figure 4 Effect diagram of the sesquiterpene compound of the present invention on the production of NO by LPS-induced BV-2; As Figure 4 shown, after LPS stimulated BV-2 cells for 24 hours, the production of NO increased significantly by about 2-fold, indicating successful modeling. After adding the sesquiterpene compound, the production of NO in the cells was significantly inhibited, indicating that the sesquiterpene compound has a strong inhibitory effect, and the inhibition rate was 56.0%.

[0082] [7] Panda K, Rosenfeld RJ, Ghosh S. 2002. Distinct dimmer interaction and regulation in nitric-oxide synthase types I, II, and III. J Biol Chem, 277: 31020 - 31030.

[0083] 1.5 Anti-inflammatory activity Western Blot Analysis:

[0084] After adding 50 μM of the sesquiterpene compound to BV2 cells and incubating for 24 hours, referring to the literature [8]The method described in [reference] was used to extract cellular proteins, and Western blotting was performed to analyze protein expression. Briefly, protein samples were boiled with loading buffer for denaturation, electrophoresed on 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked overnight at 4°C in TBST solution containing 5% BSA, and then incubated in primary antibody solutions containing iNOS, COX-2, and GAPDH (Cell signaling Technology, Boston, MA, USA) at 4°C for 4 h. After rinsing three times with TBST, the membranes were incubated in secondary antibody solution labeled with horseradish peroxidase at 4°C for 3 h. After washing the membranes, the test was performed according to the instructions of the ECL kit from GE Healthcare.

[0085] Figure 5 Figure showing the effect of the sesquiterpene compound of the present invention on the protein levels of enzyme markers in LPS-stimulated BV-2 cells; as Figure 5 shown, LPS significantly enhanced the expression of iNOS and COX-2, indicating that the sesquiterpene compound had a more obvious inhibitory effect on the expression of iNOS and COX-2, with inhibition rates of 86.4% and 85.0%, respectively.

[0086] [8] Nathan C, Xie QW. 1994. Regulation of biosynthesis of nitric oxide. J Biol Chem, 269: 13725 - 13728.

[0087] 1.6 Compounds block the nuclear subunit p65 of NF-κB in LPS-induced BV-2 cells:

[0088] NF-κB is a well-characterized transcription factor and is considered a key factor in microglia-mediated neuroinflammation. Under normal circumstances, NF-kB is bound by the inhibitory protein IκB as a p50 / p65 / IκB trimer and restricted in the cytoplasm. Once activated by various stimuli such as LPS, IκB is phosphorylated and ubiquitinated by the IκB kinase IKK, and then degraded. Therefore, the nuclear localization signal (NLS) on NF-κB is exposed, and subsequent nuclear translocation of this molecule occurs. In the nucleus, NF-κB initiates the gene transcription of related inflammatory factors [9] .

[0089] [9] Shen Qin. 2006. Mechanism of inducible nitric oxide synthase gene transcription in inflammatory glial cells. Chinese Journal of Clinical Rehabilitation, 10(30): 101 - 104.

[0090] Detection of nuclear translocation of NF-κB subunit p65 as an indicator of NF-κB activation. Cells were collected for Western blotting. To detect the nuclear translocation of NF-κB p65, a nuclear and cytoplasmic protein extraction kit was used to separate and extract the nuclear and cytoplasmic fractions according to the instructions. The primary antibodies were: anti-NF-κB p65 (1:1000), anti-Lamin B1 (1:1000) and anti-GAPDH (1:1000).

[0091] As Figure 5 shown, LPS-induced stimulation significantly increased the protein expression of phosphorylated NF-κB p65 in the nucleus, indicating that the sesquiterpene compound significantly inhibited the protein expression of phosphorylated NF-κB p65 induced by LPS, with an inhibition rate of 90.0%.

[0092] 1.7 Activity of the compound in inhibiting H2O2-induced damage in SH-SY5Y cells:

[0093] Evaluation of the protective activity against H2O2-induced oxidative stress response in neuroblastoma cell line SH-SY5Y. SH-SY5Y cells were seeded in 96-well plates at a density of 1×10 4 cells / well, and 300 μM H2O2 solution and different concentration gradients of the compound were added to each well. The cells were cultured at 37 °C under 5% CO2 for 24 h. After the drug treatment, the cell viability was measured using a CTG kit, with DMSO as the blank control, the co-treatment group of DMSO and H2O2 as the negative control group, and fluoxetine and captopril as the positive drugs. Specifically, 100 μL of CTG was added to each well, and the plate was incubated at room temperature for 10 min. The luminescence value at 570 nm was measured using a microplate reader. The data were obtained from three parallel rescreening wells and were expressed as means ± standard deviation (Means ± SD), and statistical analysis was performed using SPSS software

[10] .

[0094] Figure 6 Influence diagram of the sesquiterpene compound of the present invention on inhibiting H2O2-induced damage in SH-SY5Y cells; As Figure 6 shown, when the sesquiterpene compound was at 50 μM, it showed activity in inhibiting oxidative stress damage in SH-SY5Y cells. Compared with the damage model, its promotion rate of cell growth was 32.9%. Compared with the blank control, the cell viability was 91.6%.

[0095]

[10] Nor Hafiza Sayuti,Nabilah Zulkefii,Jen Kit Tan,Norazalina Saad,Meshari A.Alsharif,Murni Nazira Sarian.Ethanolic Extract of Polygonum minus Protects Differentiated Human Neuroblastoma Cells(SH-SY5Y)against H2O2-Induced Oxidative Stress,Molecules,2023,28,6726.

[0096] 1.8 Western Blot Analysis of Oxidative Stress:

[0097] For the detection of BCL-2 and BAX protein expression in SH-SY5Y cells by Western blot, cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1×10 5 cells per well. After 24 h of cell attachment, regular culture medium was added, and at the same time, 50 μM of the compound was added. After co-treatment for 24 h, the cells were collected. Total cell protein was extracted using a lysis buffer containing protease inhibitors, and nuclear protein was extracted using a nuclear protein extraction kit. The protein content in the tissue fluid was determined by the BCA method. Samples were loaded with 30 μg of total protein, electrophoresed on a 10% SDS-PAGE gel, and then transferred to a PVDF membrane under constant current. The membrane was blocked with 5% BSA in TBST for 1 h, and then incubated overnight at 4 °C with antibodies against BCL-2, BAX, and β-tubulin diluted 1:1000. The next day, the membrane was washed with PBST and then incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 1 h. After washing with PBST, the reagent ECL was used for development. The gray value was analyzed using Image Lab software

[11] .

[0098]

[11] Jianguo Li,Xiu Feng,Rui Ge,Jiankuan Li,Qingshan Li,Protective Effect of 2,4′,5′-Trihydroxyl-5,2′-dibromodiphenylmethanone,a New Halophenol,against Hydrogen Peroxide-Induced EA.hy926 Cells Injury.Molecules,2015,20,14254-14264.

[0099] Figure 7Effect of the sesquiterpene compound of the present invention on the protein levels of enzyme markers in H2O2-induced SH-SY5Y cells; as Figure 7 shown, H2O2 reduced cell viability, downregulated Bcl-2 expression, and upregulated Bax expression, establishing a damage model. After adding the sesquiterpene compound, the upregulation rate of Bcl-2 protein expression was 34.6%, and the downregulation rate of Bax protein expression was 27.6%, effectively preventing cell apoptosis.

[0100] In summary, the sesquiterpene compound prepared in this example can be developed as a potential lead compound and has the potential to be used as a neuroinflammation inhibitor for the treatment of neuroinflammatory diseases.

Claims

1. A sesquiterpene compound, characterized in that The chemical formula of the sesquiterpene compound is C 15 H 22 O4, and the structural formula of the sesquiterpene compound is as follows:

2. The preparation method of a sesquiterpene compound as described in claim 1, characterized in that The preparation method is carried out according to the following steps: Step 1: Inoculate Hericium erinaceus in a PDA plate medium with an inoculation amount of 5%; activate and culture at 28 °C for 7 days to obtain a well-activated fungal cake; Step 2: Inoculate the well-activated fungal cake in Step 1 onto a rice medium with an inoculation amount of 5%; culture at 28 °C for 32 days to obtain a rice fermentation medium; Step 3: Extract the rice fermentation medium in Step 2 with methanol to obtain a crude extract; extract the crude extract 3 times with a water-ethyl acetate mixed solution to obtain the extracted crude extract; fractionate with a silica gel column and sequentially use chloroform-methanol mixed solutions with volume ratios of chloroform to methanol of (100:1), (50:1), (25:1), (10:1), (5:1) and MeOH solution for gradient elution to obtain fraction a containing the target product; separate fraction a containing the target product through a chromatographic column to obtain fraction b containing the target product; purify fraction b containing the target product through a gel column, a silica gel column and a semi-preparative high performance liquid chromatograph in sequence to obtain a sesquiterpene compound.

3. The preparation method of a sesquiterpene compound according to claim 2, wherein The Hericium erinaceus described in Step 1 was purchased from the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the number 5.

579.

4. The preparation method of a sesquiterpene compound according to claim 2, characterized in that The 1000 mL PDA plate medium in Step 1 contains 10 g of peptone, 40 g of glucose and 20 g of agar powder.

5. The preparation method of a sesquiterpene compound according to claim 2, characterized in that The rice medium described in Step 2 contains 100 g of rice and 60 mL of sterile water.

6. The preparation method of a sesquiterpene compound according to claim 2, characterized in that In the water-ethyl acetate mixed solution in Step 3, water and ethyl acetate are mixed in equal volumes.

7. The preparation method of a sesquiterpene compound according to claim 2, characterized in that In Step 3, the volume ratios of the chloroform-methanol mixed solution and the MeOH solution for gradient elution are 200:200:200:100:100:100 in sequence.

8. The preparation method of a sesquiterpene compound according to claim 2, characterized in that The solvent in the chromatographic column described in Step 3 is a methanol-water mixed solution, and the elution gradient of methanol is 10% - 100%.

9. The preparation method of a sesquiterpene compound according to claim 2, characterized in that The solvent in the gel column described in Step 3 is methanol; the solvent in the silica gel column is a petroleum ether-ethyl acetate solution, and the volume ratio of petroleum ether to ethyl acetate is 9:1; the solvent in the semi-preparative high performance liquid chromatograph is a methanol-water mixed solution, the mass fraction of methanol is 40%, and the flow rate of the high performance liquid column is 2 mL / min.

10. The application of a sesquiterpene compound as described in claim 1, characterized in that The application of the sesquiterpene compound in the preparation of drugs for anti-neuroinflammation, drugs for inhibiting the production of NO by inflammatory BV2 microglia, drugs for inhibiting downstream inflammatory proteins by inhibiting the TLR4 / NF-κB signaling pathway, drugs for inhibiting the apoptotic activity of SH-SY5Y cells, and drugs for promoting Bcl-2 expression and inhibiting Bax protein expression, and the neuroinflammation includes but is not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease or Parkinson's disease.

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