Application of Stellaria dichotoma L. var. lanceolata Bge. Amine B in the preparation of anti-neuroinflammatory drugs

Abstract

The present invention discloses the application of dichotomine B in the preparation of anti-neuroinflammatory drugs, belonging to the technical field of pharmacognosy. In the early stage of the present invention, it was preliminarily confirmed that Stellaria dichotoma has anti-anxiety activity, and its active site is the n-butanol fraction, and dichotomine B (DB) was isolated therefrom. Therefore, the present invention for the first time studied the anti-inflammatory activity of DB and clarified its mechanism of action, enriched the research on the material basis of Stellaria dichotoma, and at the same time provided strong data support and reference for the development of new, highly efficient, low-toxic and low-side-effect natural anti-neuroinflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmacognosy, and more specifically, to the application of dichotomine B in the preparation of anti-neuroinflammatory drugs. Background Art

[0002] Many neurodegenerative diseases are associated with neuroinflammation, such as Alzheimer's disease, Parkinson's disease, depression, and anxiety. The neurobiological basis of these diseases has shown that stress may lead to changes in the immune system, resulting in neuroinflammation and then changes in the brain. These processes are accompanied by the continuous activation of microglia. Microglia are innate immune effector cells in the central nervous system and play a key role in maintaining tissue homeostasis, clearing pathogens, and dead cells. Activated microglia can release a variety of pro-inflammatory factors, leading to direct and indirect neurotoxic effects. Therefore, controlling microglial activation is of great significance for neuroinflammation-mediated diseases.

[0003] Lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP) stimulates BV2 microglia and ultimately affects the immune system after a series of reactions. Toll-like receptor 4 (TLR4) plays an important role in initiating the immune response. TLR4 is recognized and activated by LPS, and then myeloid differentiation factor 88 (MyD88) is activated. Finally, nuclear factor κB (NF-κB) related to inflammatory factors and mammalian target of rapamycin (mTOR) related to autophagy are activated. Related experiments have confirmed that mTOR and TLR4 play a synergistic role in regulating LPS-induced inflammatory responses. Therefore, the TLR4 / MyD88 mTOR signaling pathway plays an important role in neuroinflammation.

[0004] Stellariae Radix is the dried root of Stellaria dichotoma L. var. lanceolata Bge. of the Caryophyllaceae family. It is sweet in taste, slightly cold in nature, and belongs to the liver and stomach meridians, with the functions of clearing away deficiency-heat and eliminating infantile malnutrition-heat, and is used for yin deficiency fever, hectic fever due to yin deficiency, and infantile malnutrition-heat. Modern pharmacology has confirmed that Stellariae Radix has antipyretic, anti-inflammatory, anti-allergic, anti-cancer, vasorelaxant, relieving metabolic disorders, neuroprotective, and antioxidant activities. So far, the literature on the study of Stellariae Radix mainly focuses on the preliminary studies of the extraction process, separation and preparation, qualitative and quantitative analysis of chemical components, and pharmacological effects. Its water extracts, alcohol extracts, volatile oils, etc. have all been proven to have antipyretic and anti-inflammatory effects, and sterols, cyclic peptides, and alkaloids are considered to be the pharmacodynamic components of Stellariae Radix for anti-inflammatory and anti-cancer effects.

[0005] The research on the chemical constituents of Stellaria dichotoma var. lanceolata is still in its initial stage. The limited basic research on Stellaria dichotoma var. lanceolata has restricted the research on its mechanism of action and quality control evaluation, and also limited the rational and expanded application of resources and the development of biological resources of Stellaria dichotoma var. lanceolata. Therefore, there is an urgent need to conduct basic research on the chemical constituents of Stellaria dichotoma var. lanceolata.

[0006] In summary, how to provide an extract of Stellaria dichotoma var. lanceolata with anti-neuroinflammatory activity is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides the use of dichotomine B in the preparation of anti-neuroinflammatory drugs.

[0008] Stellaria dichotoma var. lanceolata is the dried root of the plant Stellaria dichotoma L. var. lanceolata Bge. of the Caryophyllaceae family, and is a commonly used traditional Chinese medicine in clinical practice, with the effect of clearing deficiency heat. Research has confirmed that the type of deficiency heat is related to the neuroendocrine immune network system. Preliminary studies in the early stage of the present invention have confirmed that Stellaria dichotoma var. lanceolata has anti-anxiety activity, and its active site is the n-butanol fraction, and dichotomine B (DB) has been isolated from it. Therefore, the present invention first studied the anti-inflammatory activity of DB and elucidated its mechanism of action. Finally, molecular docking technology was used to verify its activity.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The use of dichotomine B in the preparation of anti-neuroinflammatory drugs.

[0011] Furthermore, dichotomine B exerts its effect by inhibiting TLR4.

[0012] Furthermore, dichotomine B can inhibit the levels of IL-6, IL-1β and TNF-α in neuroinflammatory cells.

[0013] Furthermore, dichotomine B can exert an anti-neuroinflammatory effect by inhibiting the TLR4 / MyD88-mTOR signaling pathway and activating autophagy.

[0014] Furthermore, the preparation method of dichotomine B is as follows:

[0015] (1) Stellaria dichotoma var. lanceolata is extracted with ethanol to obtain an ethanol extract;

[0016] (2) The ethanol extract is successively extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain the n-butanol fraction;

[0017] (3) The n-butanol fraction is successively separated and purified by a silica gel column with a mesh size of 100-200 and an MCI column to obtain dichotomine B.

[0018] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects achieved by the present invention are as follows: The anti-anxiety activity of Stellaria dichotoma L. var. lanceolata Bge. was preliminarily confirmed in the early stage of the present invention, and its active site is the n-butanol fraction, and Dichotomine B (DB) was isolated therefrom. Therefore, the present invention firstly studied the anti-inflammatory activity of DB and elucidated its mechanism of action, enriching the research on the material basis of Stellaria dichotoma L. var. lanceolata Bge., and at the same time providing strong data support and reference for the development of new, highly efficient, low-toxic and low-side-effect natural anti-neuroinflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0020] Figure 1 HPLC detection result of fraction B in Example 1 of the present invention 8-3 ;

[0021] Figure 2 1H NMR spectrum of Dichotomine B (DB) in Example 1 of the present invention 1 ;

[0022] Figure 3 13C NMR spectrum of Dichotomine B (DB) in Example 1 of the present invention 13 ;

[0023] Figure 4 Chemical structure of Dichotomine B (DB) in Example 1 of the present invention

[0024] Figure 5 Docking situations of Dichotomine B and diazepam with TLR4, MyD88, and mTOR in Example 2 of the present invention. Among them, A is the docking of Dichotomine B with TLR4; B is the docking of diazepam with TLR4; C is the docking of Dichotomine B with MyD88; D is the docking of diazepam with MyD88; E is the docking of Dichotomine B with mTOR; F is the docking of diazepam with mTOR;

[0025] Figure 6Effect of Dichotomine B on the viability of BV2 cells in Example 2 of the present invention. Among them, compared with 0 μM Dichotomine B, ** P < 0.01;

[0026] Figure 7 Effect of Dichotomine B on the viability of BV2 cells induced by LPS + ATP in Example 2 of the present invention. Among them, compared with the blank group, ## P < 0.01; compared with the model group, ** P < 0.01;

[0027] Figure 8 Viability of BV2 cells in each group in Example 2 of the present invention; among them, compared with the blank group, ## P < 0.01; compared with the model group, ** P < 0.01;

[0028] Figure 9 Effect of Dichotomine B and TAK-242 on the morphology of BV2 cells induced by LPS + ATP in Example 2 of the present invention;

[0029] Figure 10 Effect of Dichotomine B and TAK-242 on the levels of cytokines IL-6 (A), IL-1β (B), and TNF-α (C) in BV2 cells induced by LPS + ATP in Example 2 of the present invention. Among them, compared with the blank group, ## P < 0.01; compared with the model group, ** P < 0.01; DB: Dichotomine B;

[0030] Figure 11 Results of Western Blot analysis in Example 2 of the present invention;

[0031] Figure 12 Effect of Dichotomine B and TAK-242 on the protein expression of p-mTOR / mTOR (A), TLR4 (B), p62 (C), Beclin-1 (D), MyD88 (E), p-RPS6 / RPS6 (F), and LC3Ⅱ / LC3Ⅰ (G) in BV2 cells induced by LPS + ATP in Example 2 of the present invention; compared with the blank group, # P < 0.05, ## P < 0.01; compared with the model group, * P < 0.05, ** P < 0.01; DB: Dichotomine B;

[0032] Figure 13 Effects of Dichotomine B and TAK-242 on the mRNA expression of mTOR (A), RPS6 (B), TLR4 (C), MyD88 (D), p62 (E), LC3B (F), and Beclin-1 (G) in LPS+ATP-induced BV2 cells in Example 2 of the present invention; compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01; DB: Dichotomine B. Detailed implementation manners

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] The medicaments required for the present invention are conventional experimental medicaments, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.

[0035] Example 1

[0036] Extraction and isolation of Dichotomine B (DB)

[0037] Stellariae Radix (42.48 Kg, collected from Tongxin County, Ningxia, China, identified by Professor Lin Dong of the School of Pharmacy, Ningxia Medical University) was added to an ultrasonic extraction tank (Beijing Dongyi Science and Technology Co., Ltd., China) in multiple batches (about 5 kg each time), and then 70% ethanol (solid-liquid ratio 1:8) was added for extraction at 65°C for 3 times, 1 h each time. After filtration, the extraction solutions were combined, concentrated by rotary evaporation, dried, weighed, and the ethanol extract was obtained and stored for later use.

[0038] The ethanol extract was suspended in an appropriate amount of distilled water and successively extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. Each solvent was extracted 3 times, and each time it was allowed to stand until the layers were clear. The extraction layers were combined and the solvents were evaporated to dryness to obtain the n-butanol fraction, which was weighed and stored for later use.

[0039] Take 295 g of the extract from the n-butanol fraction and perform preliminary separation using a silica gel chromatographic column (Qingdao Marine Chemical Factory, China) with a mesh size of 100 - 200. Gradient elution is carried out with dichloromethane - methanol (30:1, 25:1, 20:1, 15:1, 10:1, 8:1, 5:1, 1:1). Collect the eluate in bottles (250 ml), detect it using silica gel thin-layer plates, and combine similar fractions. After combination, 10 parts (B1 - B 10 ) are obtained. Fraction B8 (dichloromethane∶methanol = 5:1) is further separated and purified by MCI (Mitsubishi chemical, Japan) column chromatography, with methanol / water (1:9, 2:8, 3:7, 4:6) as the mobile phase for gradient elution. After combination, 4 parts (B 8-1 -B 8-4 ) are obtained. B 8-3 (methanol∶water = 3:7) is recrystallized into a pale yellow powder. Through HPLC detection, its purity is greater than 97%. The HPLC detection results of fraction B 8-3 are as shown in Figure 1 .

[0040] Fraction B 8-3 , a pale yellow powder, 54.98 mg, is readily soluble in DMSO, and its molecular formula is C 14 H 12 N2O4. 1 The 1H NMR and 13 13C NMR spectra are shown in Figure 2 and Figure 3 .

[0041] 1 1H NMR (400 MHz, DMSO) δ 11.75 (s, 1H, NH-9), 8.83 (s, 1H, H-4), 8.37 (d, J = 7.9 Hz, 1H, H-5), 7.73 (d, J = 8.2 Hz, 1H, H-8), 7.58 (t, J = 7.7 Hz, 1H, H-7), 7.29 (t, J = 7.5 Hz, 1H, H-6), 5.14 (t, J = 5.6 Hz, 1H, H-14), 3.87 - 3.79 (m, 2H, H-15); 13 13C NMR (101 MHz, DMSO) δ 166.60 (C-16), 145.33 (C-1), 141.08 (C-13), 135.44 (C-3), 134.96 (C-10), 128.54 (C-11), 128.50 (C-7), 121.97 (C-5), 120.76 (C-12), 120.08 (C-6), 116.12 (C-4), 112.71 (C-8), 73.75 (C-14), 65.43 (C-15). The above 11H NMR and 13 The 13C NMR spectral data are basically consistent with those reported in the literature (Sun B, Morikawa T, Matsuda H, et al. Structures of new beta-carboline-type alkaloids with antiallergic effects from Stellaria dichotoma [J]. J Nat Prod. 2004, 67(9): 1464-1469.). Therefore, the compound was identified as Dichotomine B. The chemical structure of Dichotomine B is as Figure 4 shown.

[0042] Example 2

[0043] Study on the Anti-Neuroinflammatory Activity and Mechanism of Dichotomine B in vitro Based on TLR4 / MyD88-mTOR Signaling Pathway

[0044] Statistical analysis method for the following experimental data:

[0045] GraphPad Prism 8 software (GraphPad-software Inc., CA, USA) was used for drawing, and SPSS Statistics 26 statistical software was used for data statistics. One-way analysis of variance was used for inter-group comparison, LSD test was used for variance homogeneity, and Dunnett T3 test was used for variance heterogeneity. The data are expressed as ±SD, and significant differences are indicated by #P<0.05 or *P<0.05, and extremely significant differences are indicated by ##P<0.01 or **P<0.01.

[0046] 1 Molecular docking

[0047] The structure of Dichotomine B was searched and saved using PubChem (https: / / PubChem.ncbi.nlm.nih.gov), and diazepam was used as a positive docking control. Dichotomine B was imported into Discovery Studio 2019 software (Biovia, USA) and optimized.

[0048] Search for and save the target protein structures TLR4 (2Z66), MyD88 (4DOM), and mTOR (2FAP) through the Protein Data Bank (https: / / www.rcsb.org / ), and import them into Discovery Studio 2019 software for protein modification: remove water and ligand small molecules, add hydrogen, click Clean Protein to remove redundant protein structures, define the protein as the receptor, and generate docking sites. Perform molecular docking with the target small molecule (Dichotomine B, diazepam) using LibDock, and evaluate the affinity of the molecular conformation with LibDock scores. The LibDock scores are shown in the following table:

[0049] Table 1 LibDock Scoring Results

[0050] Compound TLR4 MyD88 mTOR Dichotomine B 125.20 97.90 100.36 Positive docking control (Diazepam) 92.61 93.97 97.25

[0051] The results showed ( Figure 5 ), when Dichotomine B docked with TLR4 (2Z66), it was connected by hydrogen bonds at HIS566, ASPC596, ALAA462, and GLNA484, and the LibDock score was 125.20; when docked with MyD88 (4DOM), it was connected by hydrogen bonds at THRA185 and LEUA189, and connected by Pi-Sigma interaction at ARGA188, and the LibDock score was 97.90; when docked with mTOR (2FAP), it was connected by hydrogen bonds at TRPB2101, connected by Pi-Anion interaction at PHEB2039, and connected by Pi-Alkyl interaction at LEUB2031, and the LibDock score was 100.36. Dichotomine B showed good affinity for the above three proteins, and each score was higher than the LibDock score of the positive docking control diazepam. Thus, it can be predicted that Dichotomine B exerts anti-neuroinflammatory activity through the target proteins (TLR4, MyD88, mTOR). Based on the Libdock scores, the relative content of the isolated compounds in Stellaria dichotoma L. var. lanceolata Bge. and the characteristic components of Stellaria dichotoma L. var. lanceolata Bge., the following will study the in vitro anti-neuroinflammatory activity and mechanism of Dichotomine B based on the TLR4 / MyD88-mTOR signaling pathway.

[0052] 2 Cell Culture

[0053] Culture BV2 microglial cells (Baiyulan Institute of Pharmaceutical Research, Nanjing, China) in DMEM (Gibco, USA) high-glucose medium containing 10% fetal bovine serum (FBS) (Lonza, South America), 100 U / mL penicillin, and 100 mg / mL streptomycin (Beyotime, China) in an incubator at 37°C and 5% CO2.

[0054] 3 Determination of DB concentration

[0055] In order to evaluate whether Dichotomine B has an effect on the viability of normal cells, in this invention, the MTT method was used to treat BV2 cells with different concentrations of Dichotomine B to measure the cell survival rate.

[0056] BV2 cells in the exponential growth phase with good condition (8000 cells / well) were seeded in a 96-well plate and cultured. Different concentrations of Dichotomine B (0, 5, 10, 30, 50, 80, 100, 200 μM) were added, with 6 replicates for each concentration, and cultured for 24 h. The supernatant was discarded. 20 μL of 0.5 mg / mL MTT solution (Biosharp, China) was added to each well, and incubated at 37 °C for 4 h, then the supernatant was discarded. 150 μL of DMSO was added and shaken well to dissolve the crystals. The absorbance D(λ) of each well at 570 nm was measured with a microplate reader (Thermo Fisher, USA), and the cell survival rate was calculated.

[0057] Cell survival rate = D(λ)drug / D(λ)blank × 100%.

[0058] The results of the survival rate of BV2 cells with different concentrations of Dichotomine B are shown in Table 2, Figure 6 as follows.

[0059] Table 2 Survival rate of BV2 cells with different concentrations of Dichotomine B

[0060]

[0061]

[0062] Note: Compared with 0 μM Dichotomine B, ** P < 0.01.

[0063] The experimental results showed that compared with BV2 cells without drug intervention, Dichotomine B at 5, 10, 30, 50, 80, and 100 μM had no significant effect on the viability of BV2 cells, while Dichotomine B at 200 μM significantly decreased the survival rate of BV2 cells (P < 0.01). It was shown that Dichotomine B at 0 - 100 μM had no significant effect on promoting the proliferation or cytotoxicity of BV2 cells, and at 200 μM, Dichotomine B showed cytotoxicity to BV2 cells.

[0064] To determine the effective concentration of Dichotomine B intervention, the MTT method was used in this invention to treat BV2 cells induced by LPS + ATP with different concentrations of Dichotomine B and measure the cell viability.

[0065] BV2 cells in the exponential growth phase with good condition (8000 cells / well) were seeded in 96-well plates and cultured adherently for 24 h. The supernatant was discarded, and then the cells were treated with 10 μg / mL LPS, 5 mM ATP and different concentrations of Dichotomine B (0, 5, 10, 20, 40, 80, 100 μM) for 24 h, with 6 replicates for each concentration. After discarding the supernatant, 20 μL of 0.5 mg / mL MTT solution was added to each well and incubated in an incubator at 37 °C for 4 h, and then the supernatant was discarded. 150 μL of DMSO was added and shaken well to dissolve the crystals. The absorbance of each well was measured at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated, so as to calculate the IC 50 . The dosing concentrations were 1 / 2, 1 and 2 times of IC 50 . The results of the survival rate of BV2 inflammatory cells induced by LPS + ATP with different concentrations of Dichotomine B are shown in Table 3 and Figure 7 .

[0066] Table 3 Survival rate of BV2 inflammatory cells induced by LPS + ATP with different concentrations of Dichotomine B

[0067] Group Survival rate (%) Blank 100.00±5.28 Model <![CDATA[58.04±5.25 ## > Model + 5 μM Dichotomine B 65.15±3.56 Model + 10 μM Dichotomine B <![CDATA[68.78±5.47 ** > Model + 20 μM Dichotomine B <![CDATA[74.74±3.25 ** > Model + 40 μM Dichotomine B <![CDATA[80.72±3.52 ** > Model + 80 μM Dichotomine B <![CDATA[85.10±4.02 ** > Model + 100 μM Dichotomine B <![CDATA[88.88±5.43 ** >

[0068] Note: Compared with the blank group, ## P < 0.01; compared with the model group, ** P < 0.01.

[0069] The experimental results showed that compared with the blank group, the survival rate of BV2 cells induced by LPS + ATP was significantly decreased (P < 0.01); compared with the model group (10 μg / mL LPS + 5 mM ATP), the survival rate of model cells intervened with 10, 20, 40, 80, 100 μM of Dichotomine B was significantly increased (P < 0.01). The IC 50 value of Dichotomine B against the neuroinflammatory model cells was about 40 μM. Therefore, 20, 40 and 80 μM of Dichotomine B were selected as low, medium and high concentrations for subsequent experiments.

[0070] 4 Determination of cell viability

[0071] The experiment was divided into seven groups, namely the blank group, the model group (10 μg / mL LPS + 5 mM ATP), the model + TLR4 inhibitor group (the final concentration of TAK-242 was 10 μmol / L), the model + Dichotomine B low-dose group (the final concentration of Dichotomine B was 20 μmol / L), the model + Dichotomine B medium-dose group (the final concentration of Dichotomine B was 40 μmol / L), the model + Dichotomine B high-dose group (the final concentration of Dichotomine B was 80 μmol / L), and the Dichotomine B high-dose group (the final concentration of Dichotomine B was 80 μmol / L).

[0072] BV2 cells in the exponential growth phase with good status (8000 cells / well) were seeded in 96-well plates and cultured adherently for 24 h, then the supernatant was discarded. In the model + Dichotomine B groups at various concentrations, the Dichotomine B high-dose group, and the model + TAK-242 group, each dose of Dichotomine B or TAK-242 was added for pre-culture for 1 h, and then the supernatant was discarded. In the model group, the model + DB group, and the model + TAK-242 group, LPS (10 μg / ml) was added [not added in other groups] and continued to be cultured for 24 h, then the supernatant was discarded, and 5 mM ATP was added for treatment for 30 min, and then the culture medium was discarded. 20 μL of 0.5 mg / mL MTT solution was added to each well and incubated in an incubator at 37 °C for 4 h, then the supernatant was discarded. 150 μL of DMSO was added and shaken well to dissolve the crystals. There were 6 replicates in each group. The absorbance D(λ) of each well was measured at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0073] Cell survival rate = D(λ)drug / D(λ)blank × 100%.

[0074] The cell viability of each group is shown in Table 4 Figure 8 as follows.

[0075] Table 4 Survival rates of BV2 inflammatory cells induced by LPS + ATP resisted by Dichotomine B at various concentrations

[0076]

[0077]

[0078] Note: Compared with the blank group, ## P < 0.01; compared with the model group, ** P < 0.01.

[0079] The experimental results showed that, compared with the blank group, the cell viability of the model group was significantly decreased (P<0.01), and there was no significant difference in the cell viability of the 80 μM Dichotomine B group; compared with the model group, the cell viabilities of the model + TAK-242 group and the model + Dichotomine B group were both significantly increased (P<0.01). It was indicated that 80 μM Dichotomine B had no effect on the viability of normal BV2 cells, and high, medium, and low doses of Dichotomine B could all improve the viability of damaged cells, suggesting that Dichotomine B had anti-neuroinflammatory activity. Meanwhile, the TLR4 inhibitor TAK-242 could also improve the viability of damaged cells, preliminarily indicating that BV2 neuroinflammation might be related to the activation of TLR4, and Dichotomine B might exert anti-neuroinflammatory effects by inhibiting TLR4.

[0080] 5 Cell morphological observation

[0081] BV2 cells in the exponential growth phase with good status were seeded in 24-well plates. After adherent culture for 24 h, the supernatant was discarded. According to the experimental grouping in step 4, drugs, LPS, and ATP were added for culture, and the morphological changes of BV2 cells in each group were observed with an inverted microscope (×400) (Olympus, Japan) before and after the experiment. Each group was repeated four times.

[0082] The effects of Dichotomine B and TAK-242 on the morphology of LPS + ATP-induced BV2 cells were as Figure 9 shown.

[0083] The results showed that BV2 cells in the blank group were in a branched resting state with uniform shapes. BV2 cells in the model group had enlarged cell bodies, were round or rod-shaped, and the surface protrusions of some cells became thick and short, amoeba-like. In the model + Dichotomine B group and the model + TAK-242 group, the morphology of damaged cells was improved; the cell morphology of the 80 μM Dichotomine B group was similar to that of the blank group.

[0084] 6 Determination of cytokines by ELISA

[0085] BV2 cells in the exponential growth phase with good status (5×10 5 cells / well) were seeded in 6-well plates. After adherent culture for 24 h, according to the experimental grouping in step 4, 10 μg / mL LPS + 5 mM ATP and each drug were used for treatment for 24 h, and the supernatant was collected. The contents of IL-6, IL-1β, and TNF-α were detected with reference to the instructions of the ELISA kit (Shanghai Zhuocai Biotechnology Co., Ltd., China).

[0086] The results were as shown in Table 5, Figure 10 shown.

[0087] Table 5 Effects of Dichotomine B at various concentrations on IL-6, IL-1β, and TNF-α in LPS+ATP-induced BV2 inflammatory cells

[0088]

[0089]

[0090] Note: Compared with the blank group, ## P<0.01; compared with the model group, ** P<0.01; DB: Dichotomine B.

[0091] The results showed that compared with the blank group, the levels of IL-6, IL-1β, and TNF-α in the model group were significantly increased (P<0.01), and there was no significant difference in the levels of IL-6, IL-1β, and TNF-α in the 80 μM Dichotomine B group; compared with the model group, the levels of IL-6, IL-1β, and TNF-α in the model + Dichotomine B group and the model + TAK-242 group were significantly decreased (P<0.01). It is indicated that 80 μM Dichotomine B has no effect on the levels of related factors in normal cells, Dichotomine B can inhibit the levels of IL-6, IL-1β, and TNF-α in neuroinflammatory cells. At the same time, the trend of TAK-242 is similar to that of Dichotomine B, and Dichotomine B may inhibit the levels of the above three factors by inhibiting TLR4.

[0092] 7 Western Blot analysis

[0093] BV2 cells in the exponential growth phase with good status were selected (1×10 6Cells were seeded at a density of

[0094] (cells / well) in 6-well plates and cultured adherently for 24 h. According to the experimental grouping in step 4, the BV2 cells were treated with 10 μg / mL LPS + 5 mM ATP and various drugs for 24 h, and then the cells were collected. The cells in each treatment group were washed twice with phosphate-buffered saline (PBS) (Solarbio, China) solution, centrifuged to discard the supernatant, and RIPA lysis buffer (containing 1% PMSF, 2% phosphatase inhibitor, Beyotime, China) was added. The mixture was centrifuged at 8000 r / min at 4 °C for 10 min (Thermo Fisher, USA). The supernatant was taken, and the protein concentration of each group of samples was measured according to the instructions of the BCA protein concentration assay kit (Beyotime, China). The protein concentration of each sample group was adjusted with the original lysis buffer, and Loading buffer (5X) (Beyotime, China) was added at a ratio of 4:1, and the mixture was denatured at 100 °C for 10 min. The denatured samples were stored in a -20 °C refrigerator.

[0095] The results are shown in Figure 11 、 12 as follows.

[0096] The results showed that compared with the blank group, the protein levels of TLR4, MyD88, p-mTOR / mTOR, p62, and p-RPS6 / RPS6 in the cells of the model group were significantly up-regulated (P<0.01), while the protein levels of LC3Ⅱ / LC3Ⅰ and Beclin-1 were significantly down-regulated (P<0.01). There were no significant differences in the expression levels of each protein in the 80μM Dichotomine B group; compared with the model group, the protein levels of TLR4, MyD88, p-mTOR / mTOR, p62, and p-RPS6 / RPS6 in the cells of the model + Dichotomine B group and the model + TAK-242 group were significantly down-regulated (P<0.01, P<0.05 for p62 protein in the model + 20μM Dichotomine B group), while the protein levels of LC3Ⅱ / LC3Ⅰ and Beclin-1 were significantly up-regulated (P<0.01, P<0.05 for LC3Ⅱ / LC3Ⅰ protein in the model + 20μM Dichotomine B group). It indicated that 80μM Dichotomine B had no effect on the expression levels of related proteins in normal cells, and both Dichotomine B and TAK-242 could improve the expression levels of related proteins in neuroinflammatory cells. It was preliminarily speculated that Dichotomine B could play an anti-neuroinflammatory role by inhibiting the TLR4 / MyD88-mTOR signaling pathway and activating autophagy. The results of the model + TAK-242 group were consistent with those of the model + Dichotomine B group, further confirming that the anti-neuroinflammatory effect of Dichotomine B was related to the TLR4 / MyD88-mTOR signaling pathway and autophagy.

[0097] Analysis by 8RT-qPCR technique

[0098] BV2 cells in the exponential growth phase with good status (1×10 6 cells / well) were seeded in 6-well plates. After adherent culture for 24 h, according to the experimental grouping in step 4, after treatment with 10μg / mL LPS + 5mM ATP and each drug for 24 h, BV2 cells were collected. Total RNA of the cells in each treatment group was extracted using a total RNA extraction kit (Solarbio, China), and the concentration of total RNA in the filtrate was measured using an ultra-micro spectrophotometer. RNA was reverse transcribed into cDNA using a reverse transcription kit. Then, real-time fluorescence quantitative PCR reaction was performed using a fluorescence quantitative kit. The primer design is as follows:

[0099] Table 6 Primers

[0100]

[0101]

[0102] The results were asFigure 13 as shown

[0103] The results showed that compared with the blank group, the mRNA expressions of TLR4, MyD88, mTOR, p62, and RPS6 in the model group cells were significantly up-regulated (P<0.01), while the mRNA expressions of LC3B and Beclin-1 were significantly down-regulated (P<0.01). There were no significant differences in the mRNA expression levels in the 80 μM Dichotomine B group; compared with the model group, the mRNA expressions of TLR4, MyD88, mTOR, p62, and RPS6 in the model + 40 μM, 80 μM Dichotomine B groups, and model + TAK-242 group cells were all significantly down-regulated (P<0.01, P<0.05 for mTOR mRNA in the model + 40 μM Dichotomine B group), while the mRNA expressions of LC3B and Beclin-1 were significantly up-regulated (P<0.01). It was shown that 80 μM Dichotomine B had no effect on the mRNA expression of normal cells, and both Dichotomine B and TAK-242 could improve the mRNA expression of neuroinflammatory cells by inhibiting the TLR4 / MyD88-mTOR signaling pathway and activating autophagy. Further, the RT-qPCR technique was used to detect the expression of the above mRNAs, and the experimental results showed that the expression trends of these mRNAs were consistent with the corresponding protein expressions.

[0104] In this specification, the various embodiments are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0105] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can 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 will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of stellaria root amine B in preparing anti-neuroinflammatory drugs.

2. The application according to claim 1, characterized in that Stellaria root amine B exerts its effect by inhibiting TLR4.

3. The application according to claim 1, wherein Stellaria root amine B can inhibit the levels of IL-6, IL-1β and TNF-α in neuroinflammatory cells.

4. The application according to claim 1, wherein Stellaria root amine B can exert anti-neuroinflammatory effect by inhibiting TLR4 / MyD88-mTOR signaling pathway and activating autophagy.

5. The application according to claim 1, wherein The preparation method of stellaria root amine B is as follows: (1) Stellaria root is extracted with ethanol to obtain an ethanol extract; (2) The ethanol extract is successively extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain the n-butanol fraction; (3) The n-butanol fraction is successively separated and purified by a silica gel column with 100-200 mesh and an MCI column to obtain stellaria root amine B.

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