Use of a prenylated flavonoid compound in the preparation of an anti-inflammatory medicament

Abstract

The application belongs to the technical field of natural medicine, and provides an application of a kind of isopentenyl flavonoid compound in preparation of anti-inflammatory drug, especially in preparation of selective anti-inflammatory drug of interleukin-6. The experiment of LPS-induced RAW264.7 macrophage in vitro inflammation model proves that the compound shown in formula I can significantly inhibit the secretion of IL-6 in a dose-dependent manner, has no obvious influence on IL-1 alpha and TNF-alpha, and has high cytokine selective inhibition characteristics. The compound has no obvious toxicity to cells in the effective concentration range. The compound has the potential value of being developed as a new type of IL-6 targeted anti-inflammatory drug.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to the application of an isopentenyl flavonoid compound derived from Toona sinensis bark in the preparation of anti-inflammatory drugs, particularly in the preparation of anti-inflammatory drugs that selectively inhibit interleukin-6 (IL-6). Background Technology

[0002] Ailanthus altissima bark, the dried root bark or bark of the Ailanthus altissima (Mill.) Swingle, a plant in the Simaroubaceae family, is a traditional and commonly used medicinal material in Traditional Chinese Medicine. It possesses the effects of clearing heat and drying dampness, astringing and stopping diarrhea, and stopping bleeding. It is mainly used to treat leukorrhea, damp-heat diarrhea, hematochezia, and metrorrhagia. Modern chemical research shows that Ailanthus altissima bark contains various structural types of natural active ingredients, including quassinolides, triterpenoids, flavonoids, and alkaloids. Some of these components have been proven to have anti-inflammatory, anti-tumor, and antibacterial biological activities, indicating high medicinal development value. However, in-depth research on the activity of flavonoids in Ailanthus altissima bark, especially their selective regulation of specific pro-inflammatory cytokines, remains relatively limited.

[0003] Inflammation is a fundamental defense mechanism of the body when it is injured or invaded by pathogens. However, excessive or persistent inflammation is closely related to the occurrence and development of many diseases. Interleukin-6 (IL-6) is a key pro-inflammatory cytokine in the inflammatory response network. It is secreted by various immune cells such as macrophages, dendritic cells, and T cells, and mediates acute and chronic inflammatory responses through downstream signaling pathways such as JAK-STAT3, PI3K-Akt, and MAPK. Abnormal over-secretion of IL-6 is highly correlated with the pathological processes of a series of "IL-6-driven diseases," including cytokine release syndrome (CRS, commonly seen after CAR-T cell therapy and in severe infections), rheumatoid arthritis (RA), multicentric Castleman's disease (MCD), and age-related chronic inflammation, and is one of the important pathogenic mechanisms of these diseases.

[0004] Currently, clinical treatments targeting the IL-6 signaling pathway are mainly biologics, such as tocilizumab. While these drugs have some efficacy, they suffer from limitations including high cost, the need for intravenous or subcutaneous administration, poor patient compliance, and the potential for immunosuppression and increased infection risk with long-term use, thus failing to fully meet clinical needs. Therefore, developing small-molecule IL-6 inhibitors derived from natural products is of significant practical importance in providing novel treatment options and improving safety and ease of use. Summary of the Invention

[0005] The purpose of this invention is to provide a new use for a flavonoid compound (Formula I) derived from Toona sinensis bark, namely its use in the preparation of anti-inflammatory drugs, especially anti-inflammatory drugs that selectively inhibit IL-6, and anti-inflammatory drug compositions containing the compound.

[0006] To achieve the aforementioned objective, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of isopentenyl flavonoids as shown in Formula I in the preparation of anti-inflammatory drugs:

[0008] .

[0009] In this invention, the application specifically includes the following aspects:

[0010] (1) Application of the compound shown in Formula I in the preparation of anti-inflammatory drugs;

[0011] (2) The application of the compound shown in Formula I in the preparation of drugs that selectively inhibit the secretion of interleukin-6;

[0012] (3) The use of the compound shown in Formula I in the preparation of drugs for the prevention or treatment of IL-6-mediated inflammation-related diseases, including but not limited to cytokine release syndrome (CRS), rheumatoid arthritis, Kassman disease or aging-related chronic inflammation.

[0013] In a second aspect, the present invention provides an anti-inflammatory pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0014] The beneficial effects of adopting the above technical solution are as follows:

[0015] 1. The compound of Formula I of this invention significantly inhibited IL-6 secretion in a dose-dependent manner in an LPS-induced RAW264.7 macrophage inflammation model, while having no significant effect on IL-1α and TNF-α. This high selectivity for a single cytokine differs from the broad inhibition of the NF-κB pathway by broad-spectrum anti-inflammatory drugs such as dexamethasone, suggesting that it may target the specific upstream regulatory mechanism of IL-6 production, and has better safety expectations and novel anti-inflammatory mechanism research value.

[0016] 2. The results of the MTT assay showed that the compound of formula I had no significant cytotoxicity to RAW264.7 cells within the effective anti-inflammatory concentration range, indicating that it has a wide therapeutic window and good safety.

[0017] 3. The compound of Formula I of this invention is derived from the traditional Chinese medicine Toona sinensis bark. Toona sinensis bark is abundant and inexpensive, providing a raw material guarantee for the subsequent large-scale preparation of this compound.

[0018] 4. Based on the selective IL-6 inhibitory properties of compound I, compound I has broad clinical translational prospects in the prevention and treatment of IL-6 driven diseases such as cytokine release syndrome, rheumatoid arthritis, Castleman's disease and aging-related chronic inflammation, and is expected to become a small molecule alternative to existing biological agents for IL-6 inhibitors. Attached Figure Description

[0019] Figure 1 The image shows the HR-ESI-MS chromatogram of the compound prepared in Example 1 of this invention.

[0020] Figure 2 The ¹H-NMR spectrum (600MHz, CD3OD) of the compound prepared in Example 1 of this invention is shown.

[0021] Figure 3 The ¹³C-NMR spectrum (150MHz, CD3OD) of the compound prepared in Example 1 of this invention is shown.

[0022] Figure 4 The ¹H-¹HCOSY spectrum of the compound prepared in Example 1 of this invention;

[0023] Figure 5 The HMQC spectrum of the compound prepared in Example 1 of this invention;

[0024] Figure 6 The HMBC spectrum of the compound prepared in Example 1 of this invention;

[0025] Figure 7 The NOESY spectrum of the compound prepared in Example 1 of this invention;

[0026] Figure 8 The figure shows the effect of compound I in Example 2 of the present invention on the LPS-induced secretion levels of IL-6, IL-1α, and TNF-α in RAW264.7 cells. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0028] Example 1: Extraction of isopentenyl flavonoids

[0029] S1: The dried and pulverized Toona sinensis bark was mixed with 95% ethanol solution at a mass-volume ratio of 1 kg: 8 L, and extracted by reflux for 2 h. The extraction was repeated 3 times. The extracts were combined and concentrated under reduced pressure to obtain the extract.

[0030] S2: Disperse the extract obtained in step S1 in an appropriate amount of water, and extract it sequentially with petroleum ether and dichloromethane. Collect the dichloromethane extract and concentrate it under reduced pressure to obtain the dichloromethane extract.

[0031] S3: The dichloromethane extract obtained in step S2 was separated and purified according to the following steps to obtain the target compound:

[0032] S301: The dichloromethane extract was dissolved in dichloromethane, and an equal mass of 300-400 mesh silica gel was added and mixed. The mixture was then evaporated to dryness and separated by silica gel column chromatography at normal pressure. The dichloromethane-methanol volume ratio was eluted at a gradient of 40:1 to 2:1 to obtain a total of 10 fractions (Fr.1 to Fr.10).

[0033] S302: Separation was performed by Fr.6 atmospheric pressure silica gel column chromatography with silica gel particle size of 400-600 mesh, using a gradient elution of dichloromethane-ethyl acetate at a volume ratio of 60:1 to 3:1 to obtain 12 fractions (Fr.6-1 to Fr.6-12).

[0034] S303: Fr.6-10 was separated by medium-pressure preparative liquid chromatography with a 10%→100% methanol gradient elution at a flow rate of 40 mL / min, a detection wavelength of 210 nm, and a reference wavelength of 254 nm, yielding 12 fractions (Fr.6-10-1~Fr.6-10-12).

[0035] S304: Fr.6-10-10 was separated by high-pressure preparative liquid chromatography using an Allsphere ODS column (250×22mm, packing particle size 5μm). It was eluted isocratically with 90% acetonitrile at a flow rate of 4mL / min. The chromatographic peak at retention time tR=11.2min was collected, and the target compound was obtained by concentration under reduced pressure.

[0036] The obtained compound was a white powder, and its spectral data for HR-ESI-MS, ¹H-NMR, ¹³C-NMR, ¹H-¹HCOSY, HMQC, HMBC, and NOESY are as follows: Figures 1 to 7 As shown, the structure of the compound was confirmed by analysis of various spectra to be that of Formula I. NMR data (¹H NMR 600MHz, ¹³C NMR 150MHz, CD₃OD) are shown in Table 1.

[0037]

[0038] Example 2: Evaluation of the cytotoxicity of compound I on RAW264.7 macrophages (MTT assay)

[0039] Experimental materials: mouse mononuclear macrophage leukemia cells RAW264.7 (Shanghai Chunshi Biotechnology Co., Ltd.); DMEM high glucose medium and fetal bovine serum (Gibco); trypsin, MTT, and penicillin-streptomycin (Beyotime Biotechnology Co., Ltd.).

[0040] Experimental methods: RAW264.7 cells in logarithmic growth phase were used, and 2 × 10⁻⁶ cells were injected with the solution. 5 Cells were seeded at a density of 100 μL / well in 96-well cell culture plates and cultured at 37°C, 5% CO2 for 24 h, after which the original culture medium was removed. The drug treatment groups were treated with a solution of compound I diluted with culture medium, with final concentrations of 10, 5, 2.5, and 1.25 μmol / L, respectively. The blank control and normal control groups were treated with the corresponding solvents and culture media, with 5 replicates per group. After incubation in the 96-well plates for 48 h, 20 μL MTT solution (5 mg / mL) was added to each well under dark conditions, gently shaken to mix, and incubated for another 4 h at 37°C, 5% CO2. The supernatant was discarded, and 150 μL LDMSO was added to each well to dissolve formazan crystals. The plates were shaken for 10 min, and the optical density (OD) was measured at 570 nm using a microplate reader. Each experiment was independently repeated three times, and data processing and statistical analysis were performed using GraphPadPrism software.

[0041] Experimental results: Compound I had no significant effect on the survival rate of RAW264.7 cells within the tested concentration range (1.25–10 μmol / L), and the cell survival rate remained above 90% (1.25 μmol / L, 97.33%; 2.50 μmol / L, 97.81%; 5.00 μmol / L, 98.51%; 10.00 μmol / L, 95.01%), indicating that the compound had no significant cytotoxicity to RAW264.7 cells at effective anti-inflammatory concentrations and had good cell safety.

[0042] Example 3: Effect of Compound I of Formula I on LPS-induced secretion of inflammatory factors in RAW264.7 cells (ELISA method)

[0043] Experimental materials: Mouse mononuclear macrophage leukemia cells RAW264.7 (Shanghai Chunshi Biotechnology Co., Ltd.); DMEM high glucose medium and fetal bovine serum (Gibco); LPS (Beyotime Biotechnology Co., Ltd.); IL-6, IL-1α, TNF-α ELISA kits (Wuhan Huamei Biotechnology Co., Ltd.).

[0044] Experimental methods: RAW264.7 cells in logarithmic growth phase were used, and 2 × 10⁻⁶ cells were injected with the solution. 5Cells were seeded per well in 96-well cell culture plates (100 μL / well). The experiment was divided into a normal control group (no LPS, no drug), an LPS model group (LPS added, no drug), and drug-treated groups (LPS added, with different concentrations of drug). The final concentrations of Formula I compounds in the drug-treated groups were 10, 5, 2.5, and 1.25 μmol / L, respectively; the normal control and model groups were incubated with the corresponding solvents and culture media. After pre-incubation for 4 h, except for the normal control group, LPS (final concentration 1 μg / mL) was added to each well, and the cells were incubated at 37°C, 5% CO2 for another 24 h. The cell culture supernatant was collected, centrifuged at 3000 r / min for 20 min, and the supernatant was used. The OD values ​​were measured at 450 nm using a microplate reader according to the instructions of the respective cytokine ELISA kits, and the contents of IL-6, IL-1α, and TNF-α in each group were calculated. Each experiment was independently repeated three times, and the results were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using GraphPadPrism software.

[0045] Experimental results: such as Figure 8 As shown, compared with the normal control group, the secretion levels of IL-6, IL-1α, and TNF-α in the LPS model group were significantly increased, indicating that the LPS-induced RAW264.7 macrophage inflammation model was successfully established. Compared with the LPS model group, the secretion of IL-6 by each group of compound I showed a dose-dependent significant inhibition (p<0.0001), specifically, the inhibition rate was 93.85% at 1.25 μmol / L; 98.62% at 2.50 μmol / L; 99.39% at 5.00 μmol / L; and 99.86% at 10.00 μmol / L. There was no significant effect on the secretion levels of IL-1α and TNF-α.

[0046] The above results indicate that compound I possesses highly selective inhibitory activity against IL-6. This selectivity differs from the simultaneous inhibition of multiple inflammatory factors by broad-spectrum anti-inflammatory drugs such as dexamethasone, suggesting that compound I does not exert its anti-inflammatory effect by broadly blocking core inflammatory pathways such as NF-κB, but may instead target specific upstream signaling mechanisms related to IL-6 production and secretion. This high target selectivity not only endows it with potential specificity advantages but also provides an important lead compound foundation for exploring novel anti-inflammatory mechanisms. Clinically, there is an urgent need for selective IL-6 inhibitors in the treatment of "IL-6-driven diseases" (including CRS, rheumatoid arthritis, Castleman's disease, and age-related chronic inflammation), and the aforementioned characteristics of compound I suggest its potential application value in the treatment of these diseases.

[0047] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of isopentenyl flavonoids as shown in Formula I in the preparation of anti-inflammatory drugs: 。 2. The application according to claim 1, characterized in that, The anti-inflammatory drug is used to selectively inhibit the secretion of interleukin-6.

3. The application according to claim 1 or 2, characterized in that, The anti-inflammatory drugs are used to prevent or treat IL-6-mediated inflammation-related diseases.

4. The application according to claim 3, characterized in that, The IL-6-mediated inflammation-related diseases are selected from one or more of cytokine release syndrome, rheumatoid arthritis, Kassman disease, or age-related chronic inflammation.

5. An anti-inflammatory pharmaceutical composition, characterized in that, It contains the flavonoid compound of claim 1 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

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