CN115093388A - Flavonoid compound and preparation method and application thereof - Google Patents
Flavonoid compound and preparation method and application thereof Download PDFInfo
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- CN115093388A CN115093388A CN202210888371.2A CN202210888371A CN115093388A CN 115093388 A CN115093388 A CN 115093388A CN 202210888371 A CN202210888371 A CN 202210888371A CN 115093388 A CN115093388 A CN 115093388A
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Abstract
The invention discloses a flavonoid compound and a preparation method and application thereof, relates to the technical field of medicines, and solves the technical problem that the existing non-steroidal anti-inflammatory drug can generate more side effects after being used for a long time, wherein the flavonoid compound comprises a flavonoid compound 6-methoxy-4',5,7-trihydroxy-8- (1' - (3 '-methoxy-4' -hydroxyphenyl) ethyl) isoflavone with a brand-new framework, and the flavonoid compound is a brand-new compound extracted and separated from Sichuan blackberry lily by adopting methods such as silica gel column chromatography, SephadexLH-20, ODS, high performance liquid chromatography and the like; the flavone compound IT24 has good anti-inflammatory effect, has the characteristic of selectively inhibiting mPGES-1, has the potential of being developed into a novel anti-inflammatory drug, and provides a certain foundation for the anti-inflammatory effect of a natural compound.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a flavonoid compound and application thereof.
Background
The rhizoma Belamcandae is dry rhizome of Iridaceae Iris, and has bitter and cold nature. Has the efficacies of clearing away heat and toxic material, eliminating phlegm and relieving sore throat, and is commonly used for treating heat-toxin phlegm-fire stagnation, sore throat, abundant phlegm and saliva, cough and asthma.
Inflammation is a defensive response of the body to external stimuli or injury, manifested as redness, swelling, heat and pain. A survey by the global centers for disease burden research has shown that, from 1990 to 2017, there are approximately 36.3 billion patients with chronic inflammation. Various inflammatory diseases including Rheumatoid Arthritis (RA), Alzheimer's Disease (AD), cancer, diabetes and the like have long caused great harm to physical and mental health of patients.
Macrophages are innate immune cells that play an important role in inflammation. Macrophages can be activated by LPS, interferon-gamma (IFN-. gamma.), TNF-. alpha, extracellular matrix proteins and other chemical mediators to produce mediators of inflammationSubstances such as (NO, PGE) 2 ) And proinflammatory cytokines such as Interleukin-1 beta (Interleukin-1 beta, IL-1 beta), IL-6 and TNF-alpha. The MAPK cascade is a key pathway of cellular processes, which includes proliferation, differentiation and stress responses. It is well known that the MAPK signaling pathway comprises three important subfamilies, the extracellular-signal-regulated kinases (ERK/MAPK), the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38, which all regulate inflammatory responses. Under stimulation by LPS, MAPK pathways can be activated to secrete inflammatory mediators and proinflammatory factors. Thus, MAPK signaling pathways may serve as targets for the treatment of inflammatory diseases.
Non-steroidal anti-inflammatory drugs (NSAIDs) and Glucocorticoids (Glucocorticoids, GCs) are currently used in clinical therapy to treat inflammatory diseases. NSAIDs inhibit the production of prostaglandins by inhibiting cyclooxygenase enzymes (e.g., COX-1/COX-2). The Food and Drug Administration (FDA) and the european medicines Administration issue warnings concerning NSAIDs. Therefore, if the cyclooxygenase is not affected and other targets are acted on, the side effect can be reduced, and the cyclooxygenase can be expected to be a novel and potential therapy. Several studies have shown that inflammatory responses are associated with COX-2/mPGES-1/PGE 2 Is involved in abnormal phospholipid metabolic chains. PGE 2 Is a key mediator of inflammatory diseases. Prostaglandin synthases (PGES), including Cytosolic Prostaglandin E synthase (cPGES), mPGES-1 and Microsomal Prostaglandin E synthase 2 (mPGES-2), which are expressed in PGE 2 Play a different role in the synthesis of (a). mPGES-1 stimulates PGH under inflammatory conditions 2 Isomerization to pathological PGE 2 . At the same time, cPGES and mPGES-2 produce physiological PGE 2 . This indicates that inhibition of mPGES-1 without inhibition of upstream COX-1/COX-2 is of greater safety for treatment of inflammatory diseases.
At present, NSAIDs are clinically used for treating inflammatory diseases. However, long-term use of the composition causes more side effects, and therefore, the search for alternative anti-inflammatory drugs with less side effects is urgent.
Disclosure of Invention
The invention aims to: in order to solve the technical problem that the non-steroidal anti-inflammatory drugs can generate more side effects after being used for a long time, the invention provides a flavonoid compound and a preparation method and application thereof.
The invention specifically adopts the following technical scheme for realizing the purpose: a flavonoid compound, the structural formula of which is shown as follows:
in the technical scheme of the application: iridaceae plant rhizoma Belamcandae is a traditional Chinese medicinal material, and is commonly used for treating inflammatory diseases and hepatic diseases. Research shows that tectoridin and tectorigenin separated from rhizoma Belamcandae have 69.7% of inhibition rate on NO, and the extracted swertisin has the effect of resisting hepatitis B virus activity. In addition, the compounds in the Sichuan blackberry lily also have anticancer effect. Through apoptosis experiments, researchers find that the extracted and separated compounds 7-O-methyllaromoendandin and Iritectol B can induce apoptosis of human lung cancer cells COR-L23 in vitro. Bioactive candidate metabolites in bile, urine and excrement samples of Wistar rat orally taken by Sichuan blackberry extract are identified by mass spectrometry, and the interaction of the bioactive candidate metabolites is analyzed by network pharmacology, so that important protein targets related to cancer, cardiovascular diseases, genitourinary system diseases and digestive system diseases are found to be regulated and controlled by the metabolites. The main components of rhizoma Belamcandae are isoflavone, quinone and triterpenes. The isoflavone component is considered to be the most abundant component in the Sichuan blackberry lily and the most key component in the function. In recent years, the anti-inflammatory action of flavonoid compounds in natural plants has also been receiving attention, such as luteolin, baicalein, quercetin, and the like. The IT24 of the application is a flavone compound 6-methoxy-4',5,7-trihydroxy-8- (1' - (3 '-methoxy-4' -hydroxyphenyl) ethyl) isoflavone [6-methoxy-4',5,7-trihydroxy-8- (1' - (3 '-methoxy-4' -hydroxyphenyl) ethyl) isoflavone, IT24] with a brand-new skeleton, which is separated from Sichuan blackberry lily, and has a good anti-inflammatory effect through experiments, the characteristic of selectively inhibiting mPGES-1 is realized, and the IT24 has the potential of being developed into a novel anti-inflammatory drug, and the IT24 provides a certain foundation for the anti-inflammatory effect of a natural compound.
The anti-inflammatory effects of flavonoid compound IT24 were first discovered and demonstrated in the present application using an LPS-induced macrophage model, including (1) inhibition of inflammatory mediators (NO and PGE) in LPS-induced macrophages 2 ) And the production of pro-inflammatory factors (IL-6 and TNF-alpha); (2) in LPS-induced rat peritoneal macrophages, IT24 selectively inhibits protein and mRNA expression levels of mPGES-1, and has no inhibition effect on COX-1 and COX-2 proteins; (3) IT24 exerts an anti-inflammatory effect by inhibiting LPS-induced phosphorylation of p38/JNK protein in macrophages, and thus inhibiting the expression of mPGES-1. Therefore, IT24 may be a selective inhibitor of mPGES-1 with greater safety.
A preparation method of flavonoid compounds comprises the following steps:
and 3, performing silica gel column chromatography on the 70% ethanol elution part, separating by using dichloromethane-methanol as a mobile phase to obtain 6 parts, namely a fraction A, a fraction B, a fraction C, a fraction D, a fraction E and a fraction F, eluting by using dichloromethane-methanol, and sequentially performing silica gel column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography and high performance liquid chromatography to obtain the flavonoid IT-24.
Further, in the step 1, dried rhizoma belamcandae is taken out, crushed to 60 meshes and then extracted by 70% ethanol water under reflux for 2 times, each time for 2 hours, and the mass volume ratio of the rhizoma belamcandae to the 70% ethanol water is 1kg: 7L.
Further, in step 2, the mass of 70% ethanol water was 2.1 kg.
Further, the mass of fraction B was 580.3 g.
Further, the volume ratio of dichloromethane to methanol was 99: 1.
Further, the mobile phase of the high performance liquid chromatography is eluted by 40 percent acetonitrile water, t R ＝56～66min。
Further, the pharmaceutical composition contains the flavonoid compound or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
Further, the flavonoid compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition can be applied to preparation of medicines for treating rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, asthma, arteriosclerosis, Crohn's disease, Alzheimer's disease, Parkinson's disease, intestinal cancer, liver cancer or coronary heart disease. Among them, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, asthma, arteriosclerosis, and crohn's disease are inflammatory diseases.
Further, the flavonoid compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition is applied to preparing a medicament for inhibiting inflammation mediators and proinflammatory factors in macrophages induced by LPS (LPS), or the expression level of protein and mRNA of mPGES-1 in rat abdominal macrophages induced by LPS (LPS), or the phosphorylation of p38/JNK protein in macrophages induced by LPS, preferably, the inflammation mediators comprise NO and PGE 2 Proinflammatory factors include IL-6 and TNF- α.
The invention has the following beneficial effects:
1. the flavonoid compound 6-methoxy-4',5,7-trihydroxy-8- (1' - (3 '-methoxy-4' -hydroxyphenyl) ethyl) isoflavone [6-methoxy-4',5,7-trihydroxy-8- (1' - (3 '-methoxy-4' -hydroxyphenyl) ethyl) isoflavane, IT24] is a brand new compound extracted and separated from Sichuan blackberry lily by methods such as silica gel column chromatography, SephadexLH-20, ODS, high performance liquid chromatography and the like;
2. the flavone compound IT24 of the application remarkably inhibits NO and PGE in RAW264.7 macrophage and rat abdominal cavity macrophage induced by LPS 2 But does not affect the expression of COX-1/COX-2. In addition, IT24 selectively down-regulated LPS-induced peritoneal macrophagocytosis in ratsmPGES-1 protein expression in cells. In LPS-induced RAW264.7 cells, IT24 could not inhibit phosphorylation and nuclear translocation of NF- κ B pathway key protein, but could inhibit phosphorylation of p38/JNK protein in Mitogen-activated protein kinase (MAPK) signaling pathway. IT24 inhibits mPGES-1 expression by inhibiting p38/JNK protein phosphorylation in MAPK pathway, thereby playing a role in inhibiting inflammation;
3. the application IT24 is used as a potential mPGES-1 selective inhibitor, can be used for treating inflammatory diseases such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, asthma, arteriosclerosis, Crohn's disease and the like, Alzheimer's disease, Parkinson's disease, intestinal cancer, liver cancer and coronary heart disease, and has further research and drug development prospects;
4. LPS can bind to toll-like receptors (TLR4) on the cell membrane, leading to the activation of macrophages by activating the MAPK signaling pathway. The activated MAPK signaling pathway leads to phosphorylation of JNK, p38, and ERK, leading to subsequent transcription of inflammatory mediators and proinflammatory factors. IT24 inhibited the expression of iNOS, TNF- α, IL-6 and mPGES-1. PGE 2 Is synthesized from PGES, including mPGES-1. Thus, IT24 can inhibit PGE by reducing the content of mPGES-1 2 And (4) generating. IT24 inhibits NO and PGE by inhibiting LPS-induced expression of iNOS and mPGES-1 2 The production of (1) and the anti-inflammatory effect;
5. the IT24 of the application is a flavone compound with a brand new skeleton separated from Sichuan blackberry lily, and experiments show that the flavone compound has a better anti-inflammatory effect and the characteristic of selectively inhibiting mPGES-1, so that the invention provides a certain basis for the anti-inflammatory effect of a natural compound;
6. the anti-inflammatory effects of flavonoid compound IT24 were first discovered and demonstrated in the present application using an LPS-induced macrophage model, including (1) inhibition of inflammatory mediators (NO and PGE) in LPS-induced macrophages 2 ) And the production of pro-inflammatory factors (IL-6 and TNF- α); (2) in LPS-induced rat peritoneal macrophages, IT24 selectively inhibits protein and mRNA expression levels of mPGES-1, and has no inhibition effect on COX-1 and COX-2 proteins; (3) IT24 is obtained by inhibiting LPS-induced p38/JNK protein in macrophageThe white phosphate further inhibits the expression of mPGES-1 to play an anti-inflammatory role. Therefore, IT24 may be a selective inhibitor of mPGES-1 with greater safety.
Drawings
FIG. 1 is a graph showing the toxicity test of IT24 of the present invention on RAW264.7 cells and rat peritoneal macrophages;
FIG. 2 is the docking result of the interaction of IT24 of the present invention and prostaglandin synthetase;
FIG. 3 is a graph showing the effect of IT24 of the present invention on the expression levels of pro-inflammatory factors, inflammatory mediators, NO content, IL-6 and TNF- α proteins and mRNA expression levels in LPS-induced RAW264.7 cells and rat peritoneal macrophages;
FIG. 4 is a graph showing the effect of IT24 of the present invention on the expression levels of iNOS and COX-2 proteins and mRNA in LPS-induced RAW264.7 cells;
FIG. 5 is a graph showing the effect of IT24 of the present invention on the expression of COX-1, COX-2 and mPGES-1 proteins and mRNA expression levels in LPS-induced macrophages in rat peritoneal cavities;
FIG. 6 is a graph of the effect of IT24 of the present invention on LPS-induced expression of p65, p-p65, I.kappa.B.alpha.p-I.kappa.B.alpha.IKK.beta and p-IKK.alpha/beta proteins in RAW264.7 cells;
FIG. 7 is a graph showing the effect of IT24 of the present invention on LPS-induced nuclear translocation of NF-. kappa.B in RAW264.7 cells;
FIG. 8 is a graph showing the effect of T24 of the present invention on LPS-induced expression of p38, p-p38, ERK, p-ERK, JNK, and p-JNK proteins in RAW264.7 cells;
FIG. 9 is a molecular mechanism of the present invention IT24 for inhibiting LPS-induced macrophage activation;
FIG. 10 is a graph of mass spectral data for compound IT-24 of the present invention;
FIG. 11 is a graph of infrared data for compound IT-24 of the present invention;
FIG. 12 is a graph of UV data for compound IT-24 of the present invention;
FIG. 13 is a hydrogen nuclear magnetic resonance spectrum of compound IT-24 of the present invention;
FIG. 14 is a nuclear magnetic resonance carbon spectrum of compound IT-24 of the present invention;
FIG. 15 is a nuclear magnetic resonance DEPT-135 spectrum of compound IT-24 of the present invention;
FIG. 16 is a photograph of compound IT-24 of the present invention 1 H- 1 H COSY map;
FIG. 17 is an HSQC spectrum of compound IT-24 of the present invention;
FIG. 18 is an HMBC profile of compound IT-24 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments.
Therefore, all other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.
Example 1
As shown in fig. 1 to 7, this embodiment provides a method for preparing flavonoid compounds:
taking 20.0kg of dried rhizoma ligustici wallichii, crushing to 60 meshes, performing reflux extraction for 2 times by using 400L of 70% ethanol water, performing vacuum pressure reduction for 2 hours each time until no alcohol smell exists to obtain 2.1kg of total extract, performing D101 macroporous resin column chromatography on the total extract, and performing gradient elution by using ethanol water to obtain 40% ethanol water, 70% ethanol water (580.3g) and 95% ethanol water parts respectively. Subjecting 70% ethanol eluate to silica gel column chromatography, separating with dichloromethane-methanol as mobile phase to obtain 6 fractions (A-F), eluting fraction B (85.5g) with dichloromethane-methanol (99: 1), sequentially subjecting to silica gel column chromatography, Sephadex LH-20 column chromatography, ODS column chromatography, and high performance liquid chromatography (40% acetonitrile water eluate, t-acetonitrile, etc.) R 61min) gave compound IT-24(10 mg).
Test example 1
For the compound IT-24 prepared in example 1, the structure of the compound was determined from the physicochemical properties, ultraviolet spectrum, infrared spectrum, mass spectrum, nuclear magnetic resonance spectrum, and optical rotation spectrum (FIGS. 10 to 18).
IT-24 is a yellow amorphous powder, [ alpha ]]25.8D+4.5(c 0.95CH 3 OH)，HR-ESI-MS m/z451.1393[M+H] + (calcd. for 451.1393) (FIG. 10, mass spectrum data of compound IT-24), the molecular formula of IT-24 is deduced to be C 25 H 22 O 8 . The blue color was identified by TLC using petroleum ether and ethyl acetate (1:2) as developing agents and vanillin concentrated sulfuric acid as color developing agents.
The UV spectrum (FIG. 12, UV data of compound IT-24) shows an absorption (272nm) and the IR spectrum (FIG. 11, infrared data of compound IT-24) shows a hydroxyl group absorption (3447 cm) -1 ) Carbonyl absorption (1644 cm) -1 )。 1 H NMR spectrum (nuclear magnetic resonance hydrogen spectrum of compound IT-24 of FIG. 13) showed. delta H 8.36 is a sharp, monomodal signal, indicating that the parent nucleus of the compound is isoflavone; chemical shift δ H 7.35(2H,J＝8.8Hz),δ H 6.80(2H, J ═ 8.8Hz), each group of protons had a doublet peak indicating that the compound is ortho-coupled to the phenyl ring by 2, further indicating that the compound is a 4' -oxo-substituted isoflavonoid; delta H 13.24(1H, s) indicates the presence of a 5-OH substitution in this compound; chemical shift δ H 6.89(1H, d, J ═ 1.6Hz),6.63(1H, d, J ═ 8.0Hz),6.68(1H, dd, J ═ 8.4,1.6Hz) are the ABX system on the phenyl ring, indicating that the compound contains a 1, 3, 4 trisubstituted phenyl ring. Bonding of 13 The C NMR spectrum (the nuclear magnetic resonance carbon spectrum of the compound IT-24 in FIG. 14) and the DEPT135 spectrum (the nuclear magnetic resonance DEPT-135 spectrum of the compound IT-24 in FIG. 15) contained 3 methyl groups, 9 methine groups, and 13 quaternary carbons. Delta C 180.9, 153.9 and 121.5 are characteristic signals of the central three-carbon nucleus of the isoflavone mother nucleus. Delta C 60.2, 55.7 are two methoxy carbon signals. 1 H- 1 H COSY spectra (of compound IT-24 in FIG. 16) 1 H- 1 H COSY map) shows delta H 4.71(1H, q, J ═ 7.6Hz, H-1') and δ H 1.68(3H, d, J ═ 7.6Hz, H-2 ") is relevant, suggesting the presence of-CH-in the compound 3 A structural fragment. Comparison of IT-24 nuclear magnetic spectrum data with Compound 11(J Asian Nat pred Res.2010Nov; 12(11): 978-84.) revealed that the data with larger differences were concentrated in the B-loop part of the isoflavone mother nucleus. In HMBC profiling (HMBC profiling of compound IT-24, FIG. 18), delta H 7.35(2H, d, H-2 ', 6'), 6.80(2H, d, H-3 ', 5') and delta C 157.4 (C-4') has a distanceAs a result, it is found that C-4' on the B ring is hydroxylated. Delta H 6.89(H-2 '), 6.80 (H-5') and delta C 144.5(C-4 '), 147.1 (C-3') are remotely related, δ H 6.68 (H-6') and Δ C 144.5 (C-4') has a remote correlation, δ H 3.71(3H, s) had a remote correlation with 147.1(C-3 '") indicating that the 3'" bit was attached to-OCH 3 The 4' ″ is hydroxylated. The NMR spectroscopic data for compound IT-24 was assigned by HSQC spectroscopy (FIG. 17 HSQC spectrum of compound IT-24) as shown in Table 1. The structure of compound IT-24 is shown below:
the compound IT-24 was searched in the Scifinder database, and no relevant report was found, and IT was named 6-methoxy-4',5,7-trihydroxy-8- (1' - (3 '-methoxy-4' -hydroxyphenyl) ethyl) isovolone.
TABLE 1 NMR spectroscopic data for Compound IT-24
δinppm；J in Hz；in DMSO
Test example 2
Cytotoxicity assays
Cell culture and administration: the RAW264.7 cell line was purchased from American Type Culture Collection (ATCC) and cultured in DMEM high-glucose medium containing 10% serum (FBS). The culture conditions are constant temperature of 37 ℃ and 5% CO 2 . The treatment conditions were IT24 pre-dosed for 1h followed by stimulation with LPS (100 ng/mL).
Separating and culturing primary macrophages: rat peritoneal macrophages were isolated from SD rats (200-250g) in vivo. Isolation methods reference (biogenology. 2016apr; 17(2): 359-71). Immediately after the cells were harvested, centrifugation was carried out at 1500rpm/10min, and the supernatant was discarded and cultured in DMEM medium containing 10% FBS. Then theCells were seeded in 6-well plates at 37 ℃ with 5% CO 2 And culturing for 2 h. Different concentrations of IT24 were given for pretreatment for 1h, followed by stimulation with LPS (1. mu.g/mL).
And detecting the cell viability by adopting an MTT method, calculating the cell viability of each group according to a formula, and determining seven groups of different concentration gradients for subsequent experiments. RAW264.7 cells (1.2X 10) 4 ) And rat peritoneal macrophages (1.5X 10) 5 ) The cells were inoculated in a 96-well plate and cultured for 24 hours. Pretreatment was carried out for 1h with different concentrations of IT 24. Cells were stimulated with LPS at concentrations of 100ng/mL and 1. mu.g/mL for 24h/18h, respectively. MTT solution (5g/L) was added to each well and incubated at 37 ℃ for 4 hours. mu.L of 10% SDS-HCl solution was added to each well and incubated for 18 hours. The absorbance was read at 570nm using an ultraviolet-visible spectrophotometer. The control group was treated without any intervention and the cell viability was considered to be 100%. Cell survival (%) - (%) treatment OD value/control OD value × 100%.
MTT results showed no significant toxicity to cells in LPS-induced RAW264.7 cells when IT24<25 μ M (fig. 1A). In LPS-induced rat peritoneal macrophages, there was no significant toxicity to cells when IT24 was <50 μ M (fig. 1B). Therefore, 25 μ M and 50 μ M were selected as the highest dosing concentrations for the two types of cells, respectively.
As can be seen from fig. 1: FIG. 1(A) toxicity test of IT24 on RAW264.7 cells; IT24 (6.25-100. mu.M) pre-treated cells for 1h, and some cells were stimulated with LPS for 18h, and their cell viability was analyzed. FIG. 1(B) toxicity test of IT24 on rat peritoneal macrophages; cells were pretreated with IT24 (6.25-100. mu.M) for 1h, and a portion of the cells were stimulated with LPS for 18h, all data from three independent experiments. P <0.05, P <0.01, P <0.001, vs. normal cells or cells stimulated by LPS.
Test example 3
Molecular docking test
The Discovery Studio and PyMOL software were used for graphical editing in this experiment. COX-1, COX-2 and mPGES-1 constructs were obtained from the PubChem database and the PDB database, respectively.
The computer simulated molecular docking technology is adopted to analyze potential action targets of IT 24. IT24 may not bind directly to the active sites of COX-1 and COX-2, according to the docking scores in table 2. In contrast, IT24 has higher binding energy with the mPGES-1 target. Based on the above analysis, IT24 may not bind directly to COX-1 and COX-2, but rather has a binding effect to mPGES-1.
TABLE 2 prediction of the interaction of IT24 with prostaglandin synthetase
FIG. 2 is the docking result of the interaction of IT24 and prostaglandin synthetase; in the experiment, Discovery Studio software is used for analysis, and PyMOL software is used for graph editing.
Test example 4
ELISA and Griess experiments: RAW264.7 cells (4.5X 10) 5 ) And rat peritoneal macrophages (3X 10) 5 ) Culture in 6-well plates. The cells were pretreated with different concentrations of IT24 for 1h, stimulated with LPS at concentrations of 100ng/mL and 1. mu.g/mL for 24h/18h, respectively, and the supernatants were collected. ELISA kit is adopted to detect proinflammatory cytokines TNF-alpha and IL-6 and inflammatory mediator PGE in supernatant 2 、PGI 2 、PGD 2 And TXA 2 The level of (c). And detecting the content of NO in the supernatant by adopting a Griess kit. The real-time fluorescent quantitative reverse transcription polymerase chain reaction is adopted to detect the gene expression level of TNF-alpha and IL-6 of the cells.
The anti-inflammatory effect of IT24 was determined using Griess assay, ELISA assay, real-time fluorescent quantitative reverse transcriptase polymerase chain reaction. As shown in FIG. 3, IT24 significantly inhibited NO and PGE in LPS-induced RAW264.7 cells (FIG. 3A) and rat peritoneal macrophages (FIG. 3B) 2 The expression level of (a). In fig. 3C, IT24 significantly suppressed TXA 2 And PGD 2 Without affecting the LPS-induced PGI in rat peritoneal macrophages 2 Expression of (2). At the same time, IT24 also inhibited protein (fig. 3D) and gene (fig. 3E) expression levels of IL-6 and TNF- α in LPS-induced RAW264.7 macrophages. Based on the above results, IT24 was shown to have inhibitory effects on inflammatory mediators and proinflammatory factors in LPS-induced macrophages.
FIG. 3 (A-B) Induction of LPS by IT24Induced NO and PGE in RAW264.7 cells and rat peritoneal macrophages 2 The effect of the content; cells were pretreated with IT24 and a positive Drug (DEX) for 1h, a portion of the cells were stimulated with LPS (100 ng/mL/1. mu.g/mL) for 24h/18h, supernatants were collected, NO concentration was determined using Griess assay, PGE was measured using ELISA kit 2 The concentration of (2). (C) IT24 PGD in LPS-induced rat peritoneal macrophages 2 、PGI 2 And TXA 2 The effect of the content; (D) the effect of IT24 on TNF-. alpha.and IL-6 levels in LPS-induced RAW264.7 cells; (E) effects of IT24 on TNF-. alpha.and IL-6mRNA expression levels in LPS-induced RAW264.7 cells; the protein is measured by ELISA kit or real-time fluorescent quantitative reverse transcription polymerase chain reaction. All data were from three independent experiments. ### P<0.001, vs. Normal cells or P<0.05,**P<0.01,***P<Lps stimulated cells at 0.001, vs.
NO, as a key signaling molecule, not only causes tissue damage, but also affects the conduction of inflammatory signals in various cells and tissues in the inflammatory environment. NO, as the most important inflammatory mediator involved in the inflammatory process, can recruit leukocytes to specific tissues. Inflammatory stimuli can activate iNOS, thereby producing NO. In LPS-induced macrophages, iNOS expression was significantly increased, and IT24 of the present application showed significant inhibition of iNOS production after treatment, and thus IT24 inhibited NO production by inhibiting iNOS expression.
Prostaglandin H 2 Can be further converted into PGE 2 、PGI 2 、PGD 2 、TXA 2 And prostaglandin F 2α (prostaglandin F 2α ,PGF 2α ) Etc., thereby affecting the immune, cardiovascular, gastrointestinal and central nervous systems. mPGES-1 is the human terminal rate-limiting enzyme and was first characterized in 1999. Multiple studies have shown that aberrant expression of mPGES-1 is closely associated with various diseases. For example, mPGES-1 is overexpressed in synovial tissue samples and chondrocytes from patients with rheumatoid arthritis and osteoarthritis. We have previously found that mPGES-1 induces inflammation in the feet of rats in a Collagen-induced mouse arthritis model (Collagen-II-induced non-immune-Collagen arthritis, CIA) and in carrageenan-induced ratsOver-expression in tissues. Clinical studies found that mPGES-1 mRNA levels were reduced in Infrapatellar fat pad (IPFP) in osteoarthritis patients with hypercholesterolemia, but the effect on osteoarthritis patients after downregulation was not clear. Alzheimer disease is used as primary degenerative encephalopathy, and no specific treatment medicine for treating or reversing disease progression exists at present. Researchers find that mPGES-1 deletion can reduce accumulation of microglia around senile plaques and reduce learning disability of Tg2576 mice, and the mPGES-1 is induced in Alzheimer patients, and the novel treatment strategy for the Alzheimer patients can lay a foundation for blocking generation of mPGES-1. In addition, mPGES-1 is also involved in the pathological process of inflammatory diseases such as ankylosing spondylitis, asthma, arteriosclerosis and Crohn's disease, and diseases such as Parkinson's disease, intestinal cancer, liver cancer and coronary heart disease. Therefore, the research on how to inhibit the expression of mPGES-1 has great significance to the research progress of various diseases. Studies have shown that the PGE is a substance that acts as a carrier of 2 The key regulator produced, the selective mPGES-1 inhibitor, exerts a more beneficial cardiovascular effect than the selective COX-2 inhibitor. COX-2 as Induction PGE 2 The produced enzyme can increase PGE 2 The content of (a). The invention detects the protein expression levels of mPGES-1, COX-1 and COX-2 in macrophages in rat abdominal cavity induced by LPS. The results show that IT24 can inhibit mPGES-1 protein expression without affecting COX-1 and COX-2 expression. Until now, the mPGES-1 inhibitor GS-248 has completed phase I clinic in 2020, but GS-248 can obviously cause adverse reactions such as headache and the like. ISC 27864 has completed phase I clinic in 2015. Currently, no security and validity data for ISC 27864 at stage II is published. Candidate drugs LY3031207 and LY3023703 show selective inhibition of mPGES-1 in preclinical studies, but also cause severe adverse effects and have been terminated in phase I trials. The IT24 of the application is used as a safer potential mPGES-1 inhibitor, provides candidate drug molecules for treating inflammatory diseases such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, asthma, arteriosclerosis, Crohn's disease and the like, Alzheimer's disease, Parkinson's disease, intestinal cancer, liver cancer and coronary heart disease, and is used for treating the diseases.
Test example 5
Extracting total protein and performing Western blot experiment: protein concentrations were determined using Bio-Rad reagents. Proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to NC membranes. Placing NC membrane in 5% skimmed milk, sealing for 1h, adding TBST buffer solution, and cleaning on shaking table for 5min for three times. Primary antibody was diluted with TBST and incubated overnight in a shaker at 4 ℃. After recovery of the primary antibody, the column was washed three times for 5min each using TBST buffer on a shaker. Adding secondary antibody and incubating for 1h at room temperature in a dark place. Secondary antibody was recovered and the strips were washed three more times for 5min each with TBST buffer. The antigen-antibody complex bands were detected using the Odyssey (Li-COR, Lincoln, NE, USA) software to analyze the protein expression level.
Real-time fluorescent quantitative reverse transcription polymerase chain reaction: total RNA was isolated from RAW264.7 and rat peritoneal macrophages using the TransZol Up Plus (TransGen Biotech) RNA extraction kit. The RNA was reverse transcribed using a cDNA reverse transcription kit (Roche, Nutley, New Jersey, USA). mRNA expression levels of IL-6, TNF-alpha, iNOS and COX-2 in LPS-induced RAW264.7 cells and COX-2 and mPGES-1 in LPS-induced rat peritoneal macrophages were determined by qRT-PCR experiments. Target mRNA levels were measured by visiatm 7 real-time PCR using the FastStart Universal SYBR Green Master kit (Roche, Nutley, New Jersey, USA). Primer designs are shown in tables 3 and 4, respectively.
TABLE 3 RAW264.7 cell primers
TABLE 4 rat peritoneal macrophage primers
Effect of IT24 on iNOS and COX-2 protein and mRNA expression levels in LPS-induced RAW264.7 cells: iNOS and COX-2 are key mediators of the activation of the immune system and inflammation. Western blot and qRT-PCR experiments were used to determine the effect of IT24 on iNOS and COX-2. As shown in FIG. 4(A-C), in LPS-induced RAW264.7 cells, IT24 did not inhibit COX-2 protein expression but significantly inhibited iNOS protein expression. Furthermore, as shown in FIG. 4(D-E), IT24 inhibited the expression level of iNOS mRNA in LPS-induced RAW264.7 cells without affecting the expression level of COX-2. IT24 showed that IT could inhibit the expression of iNOS protein and mRNA but not COX-2 protein in LPS-induced RAW264.7 cells.
FIG. 4(A-C) Effect of IT24 on the expression levels of iNOS and COX-2 proteins in LPS-induced RAW264.7 cells; cells were pretreated with IT24 and a positive Drug (DEX) for 1h, and a portion of the cells were stimulated with LPS (100ng/mL) for 15 min. Total proteins of the cells were extracted and analyzed for expression levels of iNOS and COX-2 using Western blot assay. (D-E) Effect of IT24 on mRNA expression levels of iNOS and COX-2 in LPS-induced RAW264.7 cells; all data were from three independent experiments. ### P<0.001, vs. normal cells or P<0.01,***P<Lps stimulated cells at 0.001, vs.
Effects of IT24 on mPGES-1 and COX-1/COX-2 protein and mRNA expression in LPS-induced rat peritoneal macrophages: PGE can be generated under the action of mPGES-1 enzyme 2 . The downstream mPGES-1 is used as a new target point and is a safer treatment method. Western blot and qRT-PCR experiments were used to test the effect of IT24 on the expression levels of mPGES-1, COX-1 and COX-2 proteins and mRNAs in LPS-induced macrophages. As shown in FIG. 5(A-B), following LPS stimulation, mPGES-1, COX-1 and COX-2 protein expression levels were significantly up-regulated, and IT24 selectively down-regulated mPGES-1 protein expression levels, but did not inhibit COX-1 and COX-2 protein expression. The positive drug DEX also significantly down-regulated the expression of mPGES-1 and COX-2 (FIG. 5 (A-B)). The positive drug MK886 inhibits 5-lipoxygenase-activating protein (FLAP) and COX-1 activity, both inhibitors being commonly used. In this experiment, MK886 inhibited the expression level of mPGES-1 protein at a concentration of 5. mu.M/mL (FIG. 5 (A-B)). According to the qRT-PCR experimental results (FIG. 5C), IT24 at a concentration of 50. mu.M/mL significantly inhibited the mRNA expression of mPGES-1 in LPS-induced rat peritoneal macrophages. The positive drug DEX also has similar inhibitory effects on mRNA expression of COX-2 and mPGES-1. The above resultsIT24 was shown to inhibit LPS-induced mPGES-1 protein and mRNA expression in rat peritoneal macrophages without affecting COX-1 and COX-2.
FIG. 5 (A) effects of IT24 on the expression of COX-1, COX-2 and mPGES-1 proteins in LPS-induced macrophages in rat peritoneal cavity; IT24 and positive Drug (DEX) were pretreated for 1h, a portion of the cells were stimulated with LPS (1. mu.g/mL) for 18h, total cell protein was collected, and expression levels of COX-1, COX-2, and mPGES-1 were analyzed using Western blot experiments. All data were from three independent experiments. ### P<0.001, vs. Normal cells or P<0.05,**P<0.01,***P<Lps stimulated cells at 0.001, vs.
Test example 6
High content test: RAW264.7 cells (1X 10) 4 ) Inoculated in 96-well plates and cultured for 18h, and different concentrations of IT24 were pre-dosed for 6 h. In which LPS (200ng/mL) was partially administered for 15min of stimulation. Bay (1. mu.M/mL) was used as a positive drug. Nuclear translocation of NF- κ B pathway was analyzed using a high throughput inconll 6000(GE Healthcare) instrument.
Effect of IT24 on the NF-. kappa.B pathway: NF- κ B is a nuclear transcription factor that regulates inflammatory responses. The pathway can induce proinflammatory mediators such as iNOS and COX-2 and inflammatory factors such as TNF-alpha and IL-6. There are two pathways for activation of the NF-. kappa.B pathway, the classical and non-classical pathways, respectively, with the classical pathway being the most common, primarily involving the IKK β, IkappaB α and p65 proteins. Western blot experiments were used to determine the effect of IT24 on the expression levels of IKK β, p-IKK α/β, I κ B α, p-I κ B α, p65 and p-p65 proteins in LPS-induced RAW264.7 cells. As shown in FIG. 6(A-C), IT24 did not inhibit the expression of IKK- β, IkB α and p65 proteins in the NF- κ B pathway after LPS induction of RAW264.7 cells. From the above results, IT24 was shown to be unable to inhibit the activation of NF-. kappa.B pathway.
FIG. 6(A-C) Effect of IT24 on LPS-induced expression of p65, p-p65, I κ B α, p-I κ B α, IKK β and p-IKK α/β proteins in RAW264.7 cells; cells were pretreated for 1h with IT24 and positive Drug (DEX), and a portion of cells were stimulated with LPS (100ng/mL) for 15 min. Total protein from the cells was collected and analyzed by Western blot assay for expression levels of p65, p-p65, I κ B α, p-I κ B α, IKK β and p-IKK α/β. All data were from three independent experiments。 ### P<0.001, vs. normal cells or P<Lps stimulated cells, 0.01, vs.
Effect of IT24 on nuclear translocation of the NF-. kappa.B pathway: upon activation of the transcription factor complex NF-. kappa.B (p65/p50), Rel p65/p50 is released from I.kappa.B.alpha.and transported through the nuclear pore, further affecting the expression of inflammatory factors and mediators. The above results indicate that IT24 does not inhibit the activation of the NF- κ B pathway. To further validate, the effect of IT24 on LPS-induced nuclear translocation of NF- κ B in RAW264.7 cells was analyzed by high content imaging experiments. As shown in FIG. 7, IT24 did not inhibit NF- κ B nuclear translocation at any of the administered concentrations, which was consistent with the Western blot assay.
FIG. 7 effect of IT24 on LPS-induced nuclear translocation of NF-. kappa.B in RAW264.7 cells; cells were cultured in 96 wells for 18h and pretreated with varying concentrations of IT24 for 6 h. A portion of the cells were stimulated with LPS (200ng/mL) for 15 min. Nuclear translocation was analyzed using inconll 6000. ### P<0.001, vs. normal cells or P<Lps stimulated cells at 0.001, vs.
Effect of IT24 on p38/JNK protein phosphorylation expression in LPS-induced RAW264.7 cells: the MAPK signaling pathway has three important subfamilies, including JNK, ERK, and p38, which all modulate inflammatory responses. Western blot experiments were used to explore the effect of IT24 on the expression levels of p38, p-p38, ERK, p-ERK, JNK and p-JNK proteins in LPS-induced RAW264.7 cells. The results show that the expression of phosphorylated JNK, p38 and ERK proteins is remarkably enhanced after LPS stimulation. However, we found that IT24 could inhibit the expression of phosphorylated JNK and p38 proteins after predosing, but did not affect the expression of phosphorylated ERK proteins. This indicates that IT24 inhibited LPS-induced phosphorylation expression of p38/JNK protein in RAW264.7 cells.
FIG. 8. Effect of IT24 on LPS-induced expression of p38, p-p38, ERK, p-ERK, JNK, and p-JNK proteins in RAW264.7 cells; cells were pretreated with IT24 and a positive Drug (DEX) for 1h, and a portion of the cells were stimulated with LPS (100ng/mL) for 15 min. Total proteins from the cells were collected and analyzed by Western blot assay for expression levels of p38, p-p38, ERK, p-ERK, JNK, and p-JNK. ### P<0.001, vs. normal cells or P<0.05,**P<0.01,***P<Lps stimulated cells at 0.001, vs.
Proinflammatory cytokines (e.g., TNF- α and IL-6) activate neutrophils and lymphocytes, increase vascular endothelial cell permeability, regulate other tissue metabolic activities and further mediate inflammatory processes. The experimental result shows that IT24 remarkably inhibits the protein expression levels of TNF-alpha and IL-6 in RAW264.7 cells induced by LPS. In addition, NF-. kappa.B induces cytokines and adhesion molecules that regulate immune responses and recruits leukocytes to sites of inflammation. The application further explored the effect of IT24 on p65, I κ B α and IKK β protein expression. The results indicate that IT24 was unable to inhibit phosphorylation of these proteins. Meanwhile, IT24 also has no inhibitory effect on nuclear translocation of NF-kappa B p 65. Indicating that IT24 was unable to inhibit the activation of the NF-. kappa.B pathway. It is well known that MAPK also plays an important role in regulating inflammatory responses. Thus, the present application further explored the effect of IT24 on p38, JNK and ERK proteins. As shown in fig. 8, IT24 inhibited the phosphorylation expression of JNK and p38 proteins in the MAPK pathway, and had no inhibitory effect on the expression of ERK. It has been shown that subfamilies in the MAPK signaling pathway can mutually regulate mPGES-1, but the mechanism is complicated. Thus, the combined regulation of mPGES-1 and MAPK signaling pathways may be a potential approach to the treatment of inflammatory diseases, whereas IT24 may be a potent candidate drug molecule (fig. 9).
FIG. 9 molecular mechanism of IT24 inhibition of LPS-induced macrophage activation; LPS can bind to toll-like receptors (TLR4) on the cell membrane, leading to the activation of macrophages by activating the MAPK signaling pathway. Activation of the MAPK signaling pathway results in phosphorylation of JNK, p38, and ERK, leading to subsequent transcription of inflammatory mediators and proinflammatory factors. IT24 inhibited the expression of iNOS, TNF- α, IL-6 and mPGES-1. PGE 2 Is synthesized from PGES, including mPGES-1. Thus, IT24 can inhibit PGE by reducing the content of mPGES-1 2 And (4) generating. IT24 inhibits NO and PGE by inhibiting LPS-induced expression of iNOS and mPGES-1 2 Can exert anti-inflammatory effect.
In the above test examples, Lipopolysaccharide (LPS), Dexamethasone (Dexamethasone, DEX), Bay117085 and MK886 were purchased from Sigma. COX-1, COX-2, iNOS, p65, p-p65, I κ B α, p-I κ B α, IKK β, p-IKK β, p38, p-p38, ERK, p-ERK. JNK and p-JNK antibodies were purchased from CST (cell Signaling technology) Inc., USA. The β -actin and mPGES-1 antibodies were purchased from Santa (Santa Cruz), USA. P65(D14E12)
Rabbit mAb(Alexa
488Conjugate) antibody was purchased from CST corporation. Enzyme-linked immunosorbent assay kits were purchased from Cayman, usa. Griess reagent was purchased from Promega, USA.
In the above experimental examples, all data were from three independent experiments and were statistically analyzed using Graph Prism 9. Differences between groups were determined by one-way analysis of variance (ANOVA). Differences in p <0.05 are statistically significant.
Claims (10)
1. The flavonoid compound is characterized by having a structural formula as shown in the specification:
2. the preparation method of flavonoids compounds according to claim 1, characterized by comprising the following steps:
step 1, extracting Sichuan blackberry lily with ethanol, and performing vacuum pressure reduction on an extracting solution until no alcohol smell exists to obtain a total extract;
step 2, subjecting the total extract to D101 macroporous resin column chromatography, and performing gradient elution with ethanol water to obtain 40% ethanol water, 70% ethanol water and 95% ethanol water parts respectively;
and 3, performing silica gel column chromatography on the 70% ethanol elution part, separating by using dichloromethane-methanol as a mobile phase to obtain 6 parts, namely a fraction A, a fraction B, a fraction C, a fraction D, a fraction E and a fraction F, eluting by using dichloromethane-methanol, and sequentially performing silica gel column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography and high performance liquid chromatography to obtain the flavonoid IT-24.
3. The preparation method according to claim 2, wherein the dried rhizoma Belamcandae in step 1 is taken, pulverized to 60 meshes, and then extracted with 70% ethanol water under reflux for 2 times, each time for 2 hours, and the mass volume ratio of the rhizoma Belamcandae to the 70% ethanol water is 1kg: 7L.
4. The production method according to claim 2, wherein the mass of the 70% ethanol water in the step 2 is 2.1 kg.
5. The method according to claim 2, wherein the mass of fraction B is 580.3 g.
6. The method according to claim 2, wherein the volume ratio of dichloromethane to methanol is 99 to 1.
7. The method according to claim 2, wherein the HPLC mobile phase is 40% acetonitrile water elution, t R ＝56～66min。
8. A pharmaceutical composition comprising a flavonoid compound or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier.
9. Use of a flavonoid or a pharmaceutically acceptable salt thereof according to claim 1 or a pharmaceutical composition according to claim 8, preferably inflammatory mediators comprising NO and PGE, for the preparation of a medicament for inhibiting inflammatory mediators and pro-inflammatory factors in LPS-induced macrophages, or the expression levels of mPGES-1 protein and mRNA in LPS-induced rat peritoneal macrophages, or phosphorylation of p38/JNK protein in LPS-induced macrophages 2 Proinflammatory factors include IL-6 and TNF- α.
10. Use of a flavonoid compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 8 for the manufacture of a medicament for the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, asthma, arteriosclerosis, crohn's disease, alzheimer's disease, parkinson's disease, intestinal cancer, liver cancer or coronary heart disease.
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| CN1594308A (en) * | 2003-05-15 | 2005-03-16 | 成都迪康药物研究所 | Tectoridin isoflavone derivatives, their preparation process, and anti-virus medicines with tectoridin isoflavone derivatives as active components |
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