A class of lignan compounds, preparation method and use thereof

Abstract

The present invention provides a class of lignan compounds represented by the following formula, as well as their preparation methods and uses. These compounds have novel structures, can selectively and significantly inhibit the gene transcription and secretion of the cytokine IL-1β in macrophages, and exhibit excellent efficacy in treating acute liver injury at relatively low concentrations. They are expected to be used in the preparation of anti-inflammatory and hepatoprotective drugs. The present invention also relates to methods for extracting, separating, and preparing these compounds, and also to providing lead compounds for the development of anti-inflammatory and hepatoprotective drugs. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-inflammatory and hepatoprotective drugs, and specifically relates to a class of lignan compounds, preparation methods and uses thereof, and more specifically to a class of lignan compounds extracted and separated from the Euphorbiaceae plant Phyllanthus urinaria, as well as a preparation method and anti-inflammatory and hepatoprotective uses of the compounds. Background Art

[0002] Macrophages are widely distributed throughout the human body's tissues and peripheral systems, playing a crucial role in organismal development, homeostasis, tissue repair, and immune regulation. Macrophages possess remarkable plasticity, capable of polarizing into two distinct functional categories depending on the surrounding environment. Under inflammatory stimulation, macrophages polarize to a pro-inflammatory phenotype, secreting large quantities of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, to mediate tissue inflammation and defend against foreign invaders. Conversely, during the repair phase or in an anti-inflammatory setting, macrophages polarize to an anti-inflammatory phenotype, secreting anti-inflammatory cytokines that primarily participate in tissue repair and restore homeostasis. While the inflammatory response mediated by pro-inflammatory macrophages is intended to kill foreign infectants, excessive inflammatory responses can also damage the body's own tissue cells, leading to severe tissue damage and even systemic organ failure, which can be life-threatening. Inhibiting the secretion of inflammatory cytokines by macrophages can effectively block the excessive inflammatory response induced by macrophages.

[0003] Acute liver injury is a typical disease characterized by organ dysfunction or even failure caused by damage to a large number of tissue cells (hepatocytes), and has a high mortality rate. Clinically, there are many factors that lead to acute liver injury in patients: such as liver hypoxia, acute systemic inflammation, and drugs. However, inflammatory responses are involved in the pathogenesis of acute liver injury induced by these many factors. Therefore, targeted inhibition of liver inflammatory responses is expected to alleviate acute liver injury. Currently, the main clinical treatments for acute liver injury are N-acetylcysteine ​​and reduced glutathione, which are used to maintain liver homeostasis and protect the liver, but they cannot inhibit the inflammatory response at the source and prevent further liver damage. Another major class of therapeutic drugs is glucocorticoids, which are often used to inhibit systemic inflammatory responses and alleviate liver damage, but excessive use is often accompanied by numerous sequelae, which also limits their application. Therefore, it is of great significance to find drugs that target the inhibition of macrophage inflammatory responses.

[0004] Natural products are a source of anti-inflammatory drug development and lead structure discovery. According to statistics, between 2009 and 2019, nearly 321 natural products were discovered as promising anti-inflammatory drug molecules. Lignans are a diverse class of natural products, characterized by diverse structural and skeletal variations and a wide range of pharmacological activities, including antioxidant, anti-tumor, and antidiabetic activities. They are a key source of anti-inflammatory molecules.

[0005] There are over 700 species of plants in the genus Phyllanthus, Euphorbiaceae, worldwide, primarily distributed in tropical and subtropical regions, with a few in northern temperate zones. China produces 33 species and four varieties, primarily distributed in provinces and regions south of the Yangtze River. Many species of Phyllanthus, such as Phyllanthus emblica and Phyllanthus amarus, are used as folk medicinal plants in my country, for example, Phyllanthus emblica and Phyllanthus amarus, used to treat inflammatory conditions such as arthritis, conjunctivitis, vaginitis, and diarrhea. Plants in this genus also produce a rich variety of secondary metabolites with significant pharmacological activities, including immunosuppression, anti-hepatitis B virus, insect repellent, and cytotoxicity. P. franchetianus, a species of Phyllanthus, is primarily found in Sichuan and Yunnan, growing in shrubs or under sparse forests on hillsides at altitudes of 400-1000 meters.

[0006] To date, there has been no research or report on the chemical components and related anti-inflammatory pharmacological activities of Yunnan and Guizhou Leaf Urinary Pearl. Summary of the Invention

[0007] The inventors have extracted a class of lignan compounds from the aerial parts of Yunnan and Guizhou leaves for the first time and found that they have significant anti-inflammatory effects. They are a class of compound molecules with a novel skeleton and a unique anti-inflammatory mechanism, and thus have great potential uses in the development of anti-inflammatory and liver-protecting drugs.

[0008] One object of the present invention is to provide a class of lignan compounds.

[0009] Another object of the present invention is to provide a method for preparing the compound.

[0010] Another object of the present invention is to provide a pharmaceutical composition comprising the compound.

[0011] Another object of the present invention is to provide uses of the compound or its composition.

[0012] In one aspect, the present invention provides a lignan compound represented by the following formula, selected from the following compounds:

[0013]

[0014] wherein R is H, OH, OMe or OEt.

[0015] Furthermore, the lignan compound is selected from the following compounds:

[0016]

[0017]

[0018]

[0019] In the present invention, "Me" refers to methyl, "Et" refers to ethyl, "9'R" refers to the chiral carbon atom at position 9' being in R configuration, and "9'S" refers to the chiral carbon atom at position 9' being in S configuration.

[0020] In another aspect, the present invention provides a method for preparing the lignan compound, comprising the following steps:

[0021] 1) Extract the powder of the aerial part of the Yunnan Guizhou Yexiazhu with 95% ethanol aqueous solution, and evaporate the ethanol to obtain an extract;

[0022] 2) The extract was dissolved in water and extracted with ethyl acetate. The organic phase was evaporated to dryness to obtain a crude extract. The crude extract was gradient eluted on an MCI microporous resin column with a methanol-water mixture of 1:1, 3:2, 7:3, 4:1, and 9:1 by volume. The eluates from the 4:1 and 9:1 methanol-water mixtures were collected to obtain fractions F4 and F5, respectively.

[0023] 3) Component F4 in step 2) was passed through a normal phase silica gel column and eluted with a petroleum ether-acetone mixed solvent system with a volume ratio of 10:1, 5:1, 2:1, and 0:1, respectively. The eluate eluted with the petroleum ether-acetone mixed solvent with a volume ratio of 5:1 was collected to obtain component F4B; F4B was passed through a normal phase silica gel column and eluted with a petroleum ether-dichloromethane mixed solvent system with a volume ratio of 5:1, 4:1, 3:1, and 2:1, respectively. The eluate eluted with the petroleum ether-dichloromethane mixed solvent with a volume ratio of 4:1 was collected to obtain component F4B2; component F4B2 was subjected to high performance liquid chromatography and isocratically eluted with a 65% methanol-water mixed solvent to obtain compound H-2;

[0024] 4) Fraction F5 from step 2) was passed through a normal phase silica gel column and eluted with a petroleum ether-ethyl acetate mixed solvent system with a volume ratio of 1:0, 5:1, and 1:2, respectively. The eluate eluted with the petroleum ether-ethyl acetate mixed solvent with a volume ratio of 5:1 was collected to obtain fraction F5A; fraction F5A was passed through a gel column and eluted with pure methanol. The collected fractions were then subjected to high performance liquid chromatography with a gradient elution of a 60% to 90% methanol-water mixed solvent to obtain compounds H-1, H-3, and H-4, respectively.

[0025] 5) The racemic compounds H-2-H-4 obtained in steps 3) and 4) are separated by a chiral column and eluted with a mixed solvent of n-hexane-isopropanol to obtain optically pure compounds (+)-H-2 and (-)-H-2, (+)-H-3 and (-)-H-3, (+)-H-4 and (-)-H-4.

[0026] Preferably, in step 1) of the method of the present invention, the dried aerial part powder of the Yunnan Guizhou Yexiazhu is extracted with a 95% ethanol aqueous solution at room temperature three times, each time for 7 days, and the ethanol is evaporated and concentrated to obtain an extract;

[0027] Preferably, in step 5) of the method of the present invention, the chiral column is preferably an amylose or cellulose coated chiral column.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising one or more selected from the aforementioned lignan compounds, optionally and a pharmaceutically acceptable excipient.

[0029] Excipients may be pharmaceutical excipients known in the art, such as binders (such as microcrystalline cellulose), fillers (such as starch, glucose, anhydrous lactose and lactose beads), disintegrants (such as cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricants (such as magnesium stearate), as well as absorption enhancers, adsorption carriers, flavoring agents, sweeteners, excipients, diluents, wetting agents, etc.

[0030] There is no particular limitation on the dosage form of the pharmaceutical composition. It can be prepared into oral (enteral) preparations such as tablets, granules, suspensions, capsules, solutions, etc., or into parenteral preparations such as injections, etc.

[0031] In another aspect, the present invention provides use of the aforementioned lignan compound or the aforementioned pharmaceutical composition in the preparation of an anti-inflammatory drug.

[0032] In another aspect, the present invention provides use of the aforementioned lignan compound or the aforementioned pharmaceutical composition in the preparation of a drug having anti-inflammatory and hepatoprotective activities.

[0033] In another aspect, the present invention provides use of the aforementioned lignan compound or the aforementioned pharmaceutical composition in the preparation of a medicament for treating acute liver injury.

[0034] In another aspect, the present invention provides use of the aforementioned lignan compound or the aforementioned pharmaceutical composition in the preparation of a drug for specifically inhibiting the transcription and secretion of the cytokine IL-1β gene in macrophages.

[0035] The present invention relates to a class of lignan compounds with novel skeletons. In anti-inflammatory pharmacological experiments, it was found for the first time that the H-4 compound in this class can significantly and specifically inhibit the gene transcription and secretion of the pro-inflammatory cytokine IL-1β. Furthermore, it has good protective activity and efficacy against acute liver injury at low-dose administration conditions (3 mg / kg). It is inferred that this class of compounds may have preventive and therapeutic effects on a variety of inflammatory-related diseases, thus having great potential applications in the pharmaceutical field.

[0036] Therefore, the present invention provides the unique anti-inflammatory activity and mechanism of action of this type of compound, providing a basis for the development of this type of molecule into anti-inflammatory and liver-protecting drugs.

[0037] Based on the novelty of the chemical skeleton and structure and the unique mechanism of action of the anti-inflammatory and hepatoprotective activity of the lignan compound of the present invention, it has great development prospects and is expected to develop into a class of anti-inflammatory and hepatoprotective drugs with novel structure and mechanism of action.

[0038] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the above prior art or invention summary or the following examples.

[0039] Unless otherwise expressly stated, numerical ranges throughout this application include any subranges therein and any numerical values ​​in increments of the smallest subunit of a given value therein. Unless otherwise expressly stated, numerical values ​​throughout this application represent approximate measures or limits of the range of embodiments that include minor deviations from the given value and have approximately the stated value as well as the stated exact value. Except for the working examples provided at the end of the detailed description, all numerical values ​​for parameters (e.g., quantities or conditions) in this application (including the appended claims) should be understood in all cases as being modified by the term "approximately", regardless of whether "approximately" actually appears before the value. "Approximately" means that the stated value allows for slight imprecision (some approach to exactness in the value; approximately or reasonably close to the value; approximately). If the imprecision provided by "approximately" is not understood in this ordinary sense in the art, "approximately" as used herein at least represents the variation that can be produced by ordinary methods of measuring and using these parameters. For example, "approximately" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1% or less than or equal to 0.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results show that compound H-4 can reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in mouse plasma. Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0041] Figure 2 Evaluation of the inhibitory effect of compound H-4 on peripheral and liver inflammation. A: IL-1β levels in mouse plasma; B: IL-1β gene transcription levels in mouse liver tissue. Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0042] Figure 3 Evaluation of compound H-4's inhibitory effects on liver hemorrhage and hepatocyte apoptosis. A: H&E and TUNEL staining of paraffin sections of mouse liver tissue; B: Statistics of TUNEL-positive cells in panel A. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model control group. DETAILED DESCRIPTION

[0043] The following specific examples further illustrate the preparation steps and pharmacological experimental procedures of the compounds of the present invention. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and variations thereto, and the appended claims cover all such modifications within the scope of the present invention without departing from the spirit and scope of the present invention.

[0044] Example

[0045] Chemical instruments:

[0046] Optical rotation detection instrument: Autopol VI polarimeter (produced by Rudolph Technologies, USA)

[0047] UV detector: Shimadzu UV-2550 spectrophotometer (produced by Shimadzu, Japan)

[0048] Circular dichroism detector: JASCO J-810 spectrometer (manufactured by JASCO Corporation, USA)

[0049] Infrared detector: Thermo IS5 infrared spectrometer (produced by Thermo Fisher, USA)

[0050] Mass spectrometer detector: Bruker Daltonics Esquire 3000plus low-resolution mass spectrometer (manufactured by Bruker, Germany) and Waters-Micromass Q-TQF Ultima Global high-resolution mass spectrometer (manufactured by Waters, USA)

[0051] High performance liquid chromatography system: Waters 1525 system with Waters 2489 UV detector (manufactured by Waters, USA)

[0052] Pharmacology experiment related materials:

[0053] 1. Cells: The mouse macrophage cell line RAW264.7 was obtained from ATCC. Mouse bone marrow-derived primary macrophages (BMDM) were obtained from 8-week-old C57BL / 6J mice purchased from Shanghai Slack Animal Company (SLAC). Bone marrow was isolated and induced (mouse recombinant macrophage colony-stimulating factor (M-CSF, 20 ng / mL) for 7 days) and used in the experiment.

[0054] 2. Reagents:

[0055] D-galactosamine (G0500, Sigma); Lipopolysaccharides (L2630, Sigma); M-CSF (416-ML-010 / CF, R&D); DMEM-HG medium (1967442, Gibico); FBS (10091-148, Hyclone); RNAiso Plus (9109, Takara); 5X PrimerScript RT-PCR Master Mix (RR036B, Takara); 2X SYBR Green Master Mix (B21203, bimake); chloroform (Cat. No. 10006818, Sinopharm Chemical Reagent Co., Ltd.); isopropyl alcohol (Cat. No. 40064360, Sinopharm Chemical Reagent Co., Ltd.); ethanol (Cat. No. 80176961, Sinopharm Chemical Reagent Co., Ltd.); Mouse IL-1β kit (Cat. No. 62MIL1BPEG, Cisbio); ATP (Cat. No. A1852, Sigma); hydroxypropyl methylcellulose (HPMC, Cat. No. INN94653, INTECH); aspartate aminotransferase (AST) kit (Cat. Nos. R9502, R9001, Sysmex); alanine aminotransferase (ALT) kit (Cat. No. R9501, Sysmex); lactate dehydrogenase (LDH) kit (Cat. Nos. 290717, 290718, Sysmex); IL-1β enzyme-linked immunosorbent assay (ELISA) kit (Cat. No. VAL601, Novus); IN SITU CELL DEATH POD reagent (Cat. No. 11684817910, Sigma)

[0056] The sequences of the primers (synthesized by Sangon Biotech (Shanghai) Co., Ltd.) are as follows:

[0057]

[0058] 3. Consumables and instruments

[0059] 15ml centrifuge tube (Cat. No.: 430828, CORNING); 24-well cell plate (Cat. No.: 142485, THERMO) EnVision TM (Perkin Elmer); fully automatic blood parameter analyzer, SYSMEX JCA-BM6010C, Sysmex Medical Electronics (Shanghai) Co., Ltd.; optical microscope (Olympus, Tokyo, Japan).

[0060] Example 1 Preparation of the compounds of the present invention

[0061] 5 kg of dried aerial parts of the leaves of the Yunnan-Guizhou lily were extracted three times (7 days / time) with 95% ethanol and water. The mixture was condensed, refluxed, and the ethanol was evaporated to obtain an extract (350 g). The extract was dissolved in water and extracted with ethyl acetate to obtain 150 g of ethyl acetate extract. This extract was gradient eluted on an MCI microporous resin column using a methanol-water mixture with a volume ratio of 1:1, 3:2, 7:3, 4:1, and 9:1, yielding five fractions: F1 (1:1), F2 (3:2), F3 (7:3), F4 (4:1), and F5 (9:1), respectively. Fractions F4 (25 g) and F5 (35 g) from the 4:1 and 9:1 methanol-water mixtures were collected. Component F4 (25 g) was separated by a normal phase silica gel column and eluted with a petroleum ether-acetone mixed solvent system with a volume ratio of 10:1, 5:1, 2:1, and 0:1, and the eluate with a petroleum ether-acetone mixed solvent volume ratio of 5:1 was collected to obtain component F4B; F4B was separated by a normal phase silica gel column and eluted with a petroleum ether-dichloromethane mixed solvent system with a volume ratio of 5:1, 4:1, 3:1, and 2:1, and the eluate with a petroleum ether-dichloromethane mixed solvent volume ratio of 4:1 was collected to obtain component F4B2; component F4B2 was subjected to high performance liquid chromatography and isocratically eluted with a 65% methanol-water mixed solvent to obtain compound H-2. Component F5 (35 g) was separated by a normal phase silica gel column and eluted with a petroleum ether-ethyl acetate mixed solvent system with a volume ratio of 1:0, 5:1, and 1:2, and the eluate with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 5:1 was collected to obtain component F5A; component F5A was first passed through a gel column and eluted with pure methanol, and then the collected fractions were subjected to high performance liquid chromatography and gradient eluted with a 60% to 90% methanol-water mixed solvent for 30 min to obtain compounds H-1 (elution time t = 8 min peak), H-3 (elution time t = 10 min peak) and H-4 (elution time t = 16 min peak). The racemic compounds H-2-H-4 were separated by chiral column and eluted with a mixed solvent of n-hexane-isopropanol to obtain optically pure compounds (+)-H-2 and (-)-H-2, (+)-H-3 and (-)-H-3, (+)-H-4 and (-)-H-4.

[0062] Some physicochemical data of the compound are as follows:

[0063] Compound H-1: pale yellow, amorphous powder; UV (MeOH) λ max (logε)230(4.40),246(4.40),257(4.41) nm; IR(KBr)ν max 2924,2853,1690,1631,1598,1573,1494,1468,1433,1398,1337,1267,1239, 1227,1163,1131,1092,1039,933cm -1 ; (+)-ESIMS m / z 751.3[2M+Na] + ;(+)-HRESIMS m / z 751.2516[2M+Na] + (calcd for C 44 H 40 O 10 Na,751.2519).

[0064] Compound H-2: pale yellow, amorphous powder; UV (MeOH) λ max (logε)244(4.34),261(4.43)nm; IR(KBr)ν max 3408,2963,2924,2850,1731,1631,1598,1574,1494,1472,1435,1400,1341,1263, 1098,1037,804cm -1 ; (+)-ESIMS m / z 783.3[2M+Na] + ;(+)-HRESIMS m / z 783.2418[2M+Na] + (calcd for C 44 H 40 O 12 Na,783.2417).

[0065] (+)-H-2: pale yellow, amorphous powder; [α] 18 D +39.5(c 0.09,MeOH); CD(MeOH)λ max (Δε)220(+2.31),246(-0.39),259(+0.51),276(-0.63),309(+0.49),354(-0.21)nm.

[0066] (-)-H-2: pale yellow, amorphous powder; [α] 18 D -38.4(c 0.09,MeOH); CD(MeOH)λ max (Δε)219(-2.56),249(+0.24),260(-0.39),275(+0.41),309(-0.56),355(+0.11)nm.

[0067] Compound H-3: pale yellow, amorphous powder; UV (MeOH) λ max (logε)234(4.45),261(4.54)nm; IR(KBr)ν max 2930,2893,2819,1631,1600,1576,1496,1473,1433,1400,1341,1263,1239,1163, 1127,1102,1039cm -1 ; (+)-ESIMS m / z 811.3[2M+Na] + ;(+)-HRESIMS m / z 811.2726[2M+ Na] + (calcd for C 46 H 44 O 12 Na,811.2730).

[0068] (+)-H-3: light yellow, amorphous powder; [α] 17 D +5.1(c 0.13,MeOH); CD(MeOH)λ max (Δε)218(+5.96),246(-1.06),260(+0.50),275(-0.95),308(+0.83),347(-0.28)nm.

[0069] (-)-H-3: pale yellow, amorphous powder; [α] 17 D -6.9(c 0.17,MeOH); CD(MeOH)λ max (Δε)219(-3.95),245(+1.24),260(-0.39),276(+0.98),308(-0.26),344(+0.32)nm.

[0070] Compound H-4: pale yellow, amorphous powder; UV (MeOH) λ max (logε)232(4.50),261(4.60)nm; IR(KBr)νmax 2969,2927,2896,2825,1696,1629,1600,1493,1473,1437,1398,1338,1263,1234, 1163,1127,1106,1041cm -1 ; (+)-ESIMS m / z 839.3[2M+Na] + ;(+)-HRESIMS m / z839.3040[2M +Na] + (calcd for C 48 H 48 O 12 Na,839.3043).

[0071] (+)-H-4: pale yellow, amorphous powder; mp 154-156°C; [α] 16 D +23.9(c 0.17,MeOH); CD(MeOH)λ max (Δε)217(+6.50),245(-0.66),259(+2.00),276(-1.17),308(+0.74),348(-0.46) nm.

[0072] (-)-H-4: pale yellow, amorphous powder; [α] 17 D -20.0(c 0.12,MeOH); CD(MeOH)λ max (Δε)217(-6.97),246(+0.62),261(-1.60),276(+0.91),308(-0.78),348(+0.46)nm.

[0073] Example 2 Evaluation of the in vitro anti-inflammatory and in vivo hepatoprotective activities of the compounds of the present invention

[0074] In vitro anti-inflammatory activity evaluation:

[0075] When macrophages are stimulated by the endotoxin lipopolysaccharide (LPS), they activate downstream transcription factors such as NF-κB through Toll-like receptor 4 (TLR4) on their cell surface, leading to a significant upregulation of the transcriptional levels of inflammatory genes such as IL-1β, IL-6, and TNF-α. Measuring the transcriptional levels of these pro-inflammatory genes can reflect the activation state of macrophages. Unlike inflammatory factors such as IL-6 and TNF-α, IL-1β in macrophages is first translated and synthesized as an inactive IL-1β precursor (pro-IL-1β) under LPS induction. Subsequently, after ATP and other factors induce the assembly and activation of the NLRP3 inflammasome, caspase1 undergoes self-cleavage activation (forming cleaved caspase1). Cleaved caspase1 further cleaves pro-IL-1β into its active form (cleaved IL-1β), which is then secreted and exerts its effects. Under LPS and ATP induction, IL-1β levels in the cell supernatant are measured to assess the effects of compounds on macrophage IL-1β secretion. The mouse-derived macrophage cell line RAW264.7 and mouse bone marrow-derived primary macrophages BMDM are commonly used cell systems for evaluating macrophage activation and function in vitro. In the present invention, they are used to evaluate the effects of compounds on the transcription of pro-inflammatory genes and the secretion of IL-1β, respectively.

[0076] Preparation of test samples: The compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution.

[0077] 1) RAW264.7 cells in the logarithmic growth phase were seeded in 48-well plates and cultured overnight. The compounds (10 μM, 20 μM) were added. A negative control group and a model control group containing the same concentration of DMSO were set up at the same time. Each group had 3 replicates and cultured in a 37°C, 5% CO2 incubator for 4 hours. Except for the negative control group, LPS (100 ng / mL) was added to the other groups and induced for another 2 hours. The cell culture medium was then discarded, and total cell RNA was extracted and converted into cDNA. The transcription levels of pro-inflammatory genes IL-1β, IL-6, and TNF-α were detected by RT-qPCR. -ΔΔCt The data were processed by the method, and the expression levels of the model control group (C) and the compound group (T) were calculated respectively. The transcription inhibition rate was calculated using the following formula: inhibition rate (%) = [(CT) / C]×100.

[0078] 2) After BMDM cells seeded in 48-well plates were induced to mature, the compounds (10 μM, 20 μM) were added. A negative control group and a model control group containing the same concentration of DMSO were set up at the same time, with three replicates for each group. After culturing for 2 hours in a 37°C, 5% CO2 incubator, LPS (100 ng / mL) was added to the remaining groups except the negative control group and induced for another 4 hours. ATP (2 mM) was then added for induction for 45 minutes. The cell supernatant was collected for detection of IL-1β concentration. The IL-1β concentration (C) of the model control group and the IL-1β concentration (T) of the compound group were measured, and the secretion inhibition rate was calculated using the following formula: Inhibition rate (%) = [(CT) / C] × 100.

[0079] Table 1. Test results of the effects of the compounds of the present invention on the transcription levels of pro-inflammatory genes IL-1β, IL-6, and TNF-α in macrophages

[0080]

[0081]

[0082] Note: Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0083] Test results: As can be seen from the test results (Table 1), at a concentration of 20 μM, compounds (-)-H-3, (+)-H-3, (-)-H-4, and (+)-H-4 significantly inhibited IL-1β transcription; (-)-H-4 and (+)-H-4 were equally active and more potent than (-)-H-3 and (+)-H-3; whereas H-1, (-)-H-2, and (+)-H-2 had no significant effect on IL-1β transcription levels; and none of the tested compounds had a significant inhibitory effect on IL-6 or TNF-α transcription levels. This indicates that (-)-H-4 and (+)-H-4 can significantly and specifically inhibit the transcription of the pro-inflammatory gene IL-1β in macrophages.

[0084] Table 2. Test results of the effects of the compounds of the present invention on the secretion level of IL-1β in macrophages

[0085]

[0086] Note: Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0087] Test Results: As can be seen from the test results (Table 2), compounds H-1 (20 μM), (+)-H-2, (+)-H-3 (20 μM), (-)-H-3 (20 μM), (+)-H-4, and (-)-H-4 (20 μM) can significantly downregulate IL-1β secretion levels, and (-)-H-4 and (+)-H-4 are more active than the other compounds, while (-)-H-2 has no significant effect on IL-1β secretion levels. This indicates that (-)-H-4 and (+)-H-4 can significantly inhibit IL-1β secretion in macrophages.

[0088] In vivo hepatoprotective activity evaluation:

[0089] LPS (Lipopolysaccharide) combined with D-GalN (D-galactosamine) can induce an acute inflammatory response in the liver, leading to massive hepatocyte death and liver dysfunction. This is a classic experimental animal model of acute liver injury, often used to evaluate macrophage activation and pro-inflammatory cytokine production under liver injury conditions.

[0090] Preparation of test compound H-4 (racemic form): The compound was dissolved in a solvent (DMSO: 0.5% hydroxypropyl methylcellulose (HPMC) = 10:90, v / v) to prepare a 0.3 mg / mL suspension. Preparation of modeling drugs: A mixed solution containing LPS (2 μg / mL) and D-GalN (40 mg / mL) was prepared in physiological saline (0.9% NaCl). 30 male C57BL / 6J mice, 8-9 weeks old, were randomly divided into 3 groups: blank control group, model control group, and compound H-4. The 3 mg / kg group was given the compound three times in advance, once a day, by oral gavage (0.1 mL / 10 g). The blank control group and the model control group were given the corresponding solvent at the same dosage and volume. 15 minutes after the last dose, the model drug solution was administered by intraperitoneal injection (0.1 mL / 10 g, i.e., LPS (20 μg / kg) + D-GalN (400 mg / kg)). 5 hours after modeling, blood was collected from the orbits, and plasma was centrifuged and cryopreserved. The plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and IL-1β were measured. The liver was removed and the corresponding indicators of liver bleeding, inflammation, and cell apoptosis were detected (H&E staining, TUNEL-deoxyribonucleotidyl transferase-mediated nick end labeling staining, and the transcription level of the pro-inflammatory gene IL-1β - the blank control group was set as 1, and the fold change of each group relative to the blank control group was calculated). Figure 1The results show that compound H-4 can reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in mouse plasma. Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0091] Figure 2 Evaluation of the inhibitory effect of compound H-4 on peripheral and liver inflammation. A: IL-1β levels in mouse plasma; B: IL-1β gene transcription levels in mouse liver tissue. Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0092] Figure 3 Evaluation of compound H-4's inhibitory effects on liver hemorrhage and hepatocyte apoptosis. A: H&E and TUNEL staining of paraffin sections of mouse liver tissue; B: Statistics of TUNEL-positive cells in panel A. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model control group.

[0093] Test results: From the test results, it can be seen that in the model control group, mice showed obvious liver damage, which led to a significant increase in the levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase in the plasma. Compound H-4 administration can significantly reduce the above indicators ( Figure 1 At the same time, the plasma inflammatory factor IL-1β content in the H-4 administration group was significantly reduced ( Figure 2 Middle A), the transcription level of pro-inflammatory gene IL-1β in liver tissue of H-4 administration group was significantly downregulated ( Figure 2 Middle B); H&E staining analysis showed that the liver bleeding in the H-4 group was significantly improved ( Figure 3 Middle A), TUNEL staining analysis showed that the level of liver cell apoptosis in the H-4 administration group was significantly alleviated ( Figure 3 Middle A, Figure 3 (B, B is the statistical graph of TUNEL staining in Figure A).

[0094] Test results indicate that compound H-4 significantly and specifically inhibits the gene transcription and secretion of the pro-inflammatory cytokine IL-1β, and exhibits excellent protective activity and efficacy against acute liver injury at a relatively low dose (3 mg / kg). Therefore, this compound has promising development prospects in the preparation of drugs for the treatment of certain anti-inflammatory diseases and is expected to become a novel anti-inflammatory and hepatoprotective drug formulation. SEQUENCE LISTING <110> Shanghai Institute of Materia Medica, Chinese Academy of Sciences <120> DI22-0110-XC37 <130> A class of lignan compounds, preparation method and use thereof <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Forward primer <400> 1 tggtaccaca ggcattgtga t 21 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 2 tgatgtcacg cacgatttcc ct 22 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Forward primer <400> 3 gaagttccca aatggcctcc 20 <210> 4 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 4 ttgtcactcg aattttgaga agatg 25 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer <400> 5 gcaactgttc ctgaactcaa ct 22 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 6 atcttttggg gtccgtcaac t 21 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Forward primer <400> 7 tagtccttcc taccccaatt tcc 23 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 8 ttggtcctta gccactcctt c 21

Claims

1. Lignan compounds represented by the following formula: in, R is H, OH, OMe or OEt.

2. The lignan compound according to claim 1, selected from the following compounds:

3. The method for preparing the lignan compound according to claim 2, comprising the following steps: 1) Extract the powder of the aerial part of the Yunnan Guizhou Yexiazhu with 95% ethanol aqueous solution, and evaporate the ethanol to obtain an extract; 2) The extract was dissolved in water and extracted with ethyl acetate. The organic phase was evaporated to dryness to obtain a crude extract. The crude extract was gradient eluted on an MCI microporous resin column with a methanol-water mixture of 1:1, 3:2, 7:3, 4:1, and 9:1 by volume. The eluates from the 4:1 and 9:1 methanol-water mixtures were collected to obtain fractions F4 and F5, respectively. 3) Component F4 in step 2) was passed through a normal phase silica gel column and eluted with a petroleum ether-acetone mixed solvent system with a volume ratio of 10:1, 5:1, 2:1, and 0:1, respectively. The eluate eluted with the petroleum ether-acetone mixed solvent with a volume ratio of 5:1 was collected to obtain component F4B; F4B was passed through a normal phase silica gel column and eluted with a petroleum ether-dichloromethane mixed solvent system with a volume ratio of 5:1, 4:1, 3:1, and 2:1, respectively. The eluate eluted with the petroleum ether-dichloromethane mixed solvent with a volume ratio of 4:1 was collected to obtain component F4B2; component F4B2 was subjected to high performance liquid chromatography and isocratically eluted with a 65% methanol-water mixed solvent to obtain compound H-2; 4) Fraction F5 from step 2) was passed through a normal phase silica gel column and eluted with a petroleum ether-ethyl acetate mixed solvent system with a volume ratio of 1:0, 5:1, and 1:2, respectively. The eluate eluted with the petroleum ether-ethyl acetate mixed solvent with a volume ratio of 5:1 was collected to obtain fraction F5A; fraction F5A was passed through a gel column and eluted with pure methanol. The collected fractions were then subjected to high performance liquid chromatography with a gradient elution of a 60% to 90% methanol-water mixed solvent to obtain compounds H-1, H-3, and H-4, respectively. 5) The racemic compounds H-2-H-4 obtained in steps 3) and 4) are separated by a chiral column and eluted with a mixed solvent of n-hexane-isopropanol to obtain optically pure compounds (+)-H-2 and (-)-H-2, (+)-H-3 and (-)-H-3, (+)-H-4 and (-)-H-4.

4. The method for preparing the lignan compound according to claim 3, in, In step 1), the dried aerial part powder of the Yunnan Guizhou Yexiazhu was extracted with a 95% ethanol aqueous solution at room temperature three times, each time for 7 days, and the ethanol was evaporated and concentrated to obtain an extract; In step 5), the chiral column is an amylose or cellulose coated chiral column.

5. A pharmaceutical composition comprising one or more lignan compounds selected from the group consisting of claim 1 or 2, and optionally, pharmaceutically acceptable excipients.

6. Use of the lignan compound according to claim 1 or 2 or the pharmaceutical composition according to claim 5 in the preparation of anti-inflammatory drugs.

7. Use of the lignan compound according to claim 1 or 2 or the pharmaceutical composition according to claim 5 in the preparation of a medicament having anti-inflammatory and hepatoprotective activities.

8. Use of the lignan compound according to claim 1 or 2 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating acute liver injury.

9. Use of the lignan compound according to claim 1 or 2 or the pharmaceutical composition according to claim 5 in the preparation of a drug for specifically inhibiting the transcription and secretion of the cytokine IL-1β gene in macrophages.

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