A diterpenoid compound inhibiting nlrp3 inflammasome and application thereof

Abstract

The application belongs to the field of medicine, and particularly relates to a diterpene compound for inhibiting NLRP3 inflammasome and application thereof. A new compound is separated from naked flower purple perilla, and is identified as a diterpene. Cell experiments show that the compound can significantly inhibit the expression of NLRP3 inflammasome in LPS-induced human monocyte cell line THP-1 derived macrophages, and also has a significant inhibitory effect on the expression of IL-1beta and IL-18. These results show that the compound has significant anti-inflammatory activity, and provides a candidate compound for the research and development of anti-inflammatory drugs, and has important potential application value in the treatment of skin wound healing and other skin wound inflammatory diseases, diseases or pathological processes accompanied by inflammation.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a diterpenoid compound that inhibits the NLRP3 inflammasome and its applications. Background Technology

[0002] NLRP3 (NOD, LRR, and PYD domain protein 3) belongs to the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family and is one of the most thoroughly studied core components of the inflammasome. The NLRP3 inflammasome is a key component of the innate immune system, serving as a platform for pro-inflammatory cytokine secretion and playing a central regulatory role in wound healing. When wound tissue cells activate NLRP3 via Toll-like receptors and mitochondria, the NF-κB pathway is activated, leading to upregulation of inflammasome-related proteins (such as IL-1β and IL-18) expression and hindering healing. This activation process also triggers the aggregation of inflammasome sensor proteins and adaptor proteins, recruits and activates Caspase-1, promotes IL-1β / IL-18 maturation, and induces pro-inflammatory cell death. By regulating the NLRP3 inflammasome pathway, macrophage infiltration can be inhibited, angiogenesis promoted, and IL-1β secretion reduced, thereby improving cellular hypoxia response, regulating cell proliferation and differentiation, and accelerating wound repair. Therefore, inhibiting this pathway is an important strategy for treating wound healing.

[0003] Chronic inflammation is closely related to arthritis, neurodegenerative diseases, and other conditions. Long-term uncontrolled inflammation can induce DNA damage and even cancer. Regulating the inflammatory response is key to disease treatment. The NLRP3 inflammasome is an important target for drug development, and its inhibitors have potential value in the prevention and treatment of NLRP3-related diseases. Exploring natural drug resources to develop highly effective inhibitors is of great significance.

[0004] Callicarpa nudiflora ( Callicarpa nudiflora Hook. et Arn.) belongs to the genus Callicarpa in the family Verbenaceae ( CallicarpaThe plant *Callicarpa nudiflora* (L.) possesses antibacterial, hemostatic, anti-inflammatory, detoxifying, and swelling-reducing properties. It is primarily used to treat suppurative inflammation, acute infectious hepatitis, respiratory and digestive tract bleeding, and traumatic bleeding, and can be used for various acute and chronic inflammations. Externally, it is used to treat burns, scalds, and external bleeding. Clinical studies show that *Callicarpa nudiflora* has good anti-infective effects in burn treatment; it has strong astringent properties, controlling the extravasation of body fluids from the wound; and it promotes epithelial growth and accelerates wound healing; it has no significant local or systemic toxicity. Therefore, the discovery of an active ingredient in *Callicarpa nudiflora* that inhibits the NLRP3 inflammasome provides a scientific basis for revealing the anti-inflammatory material basis and mechanism of action of *Callicarpa nudiflora*, and provides a research foundation for the study of the lead compound of *Callicarpa nudiflora* that inhibits the NLRP3 inflammasome. Summary of the Invention

[0005] The technical solution of this invention is implemented as follows: A diterpenoid compound, the structural formula of which is shown in Formula I: .

[0006] The extraction method for the diterpenoid compound includes the following steps: (1) The dried leaves of Callicarpa nudiflora were extracted by heating and reflux with 95% ethanol. After the ethanol was recovered, the leaves were dissolved in water and then extracted with dichloromethane to obtain the dichloromethane extract. (2) The dichloromethane extract was separated by silica gel column chromatography and the eluent was obtained by gradient elution with a mixture of petroleum ether and ethyl acetate. (3) The fraction obtained by the volume ratio of petroleum ether and ethyl acetate of 100:10 was separated by silica gel column chromatography and eluted by gradient with a mixed solution of petroleum ether and ethyl acetate. The fraction eluted by the volume ratio of petroleum ether and ethyl acetate of 10:1 was further separated and purified by semi-preparative high performance liquid chromatography to obtain compound I.

[0007] Furthermore, in steps (2) and (3), the silica gel used in the silica gel column is 200-300 mesh; in step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 100:0-0:100; in step (3), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 40:1-0:100.

[0008] Further, in step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 100:0, 100:1, 100:2, 100:4, 100:5, 100:8, 100:10, 100:12, 100:20, 100:50, 0:100 respectively; in step (3), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 40:1, 30:1, 25:1, 20:1, 15:1, 12:1, 10:1, 8:1, 5:1, 1:1, 0:1 respectively; in step (3), during the semi-preparative high performance liquid chromatography separation process, the eluent is methanol-0.2% formic acid water = 73:27, the flow rate is 2 mL / min, and the retention time of the compound of formula I is 58 minutes.

[0009] Furthermore, in step (3), the column used in the semi-preparative high-performance liquid chromatography is a YMC-pack ODS-Acolumn with a column length of 250 mm, an inner diameter of 10 mm, a particle size of 5 μm, and a micropore size of 12 nm. The manufacturer is YMCCo., Ltd., Kyoto, Japan.

[0010] The application of the diterpenoid compound in the preparation of anti-inflammatory drugs.

[0011] The application of the diterpenoid compound in the preparation of drugs that promote wound healing.

[0012] The use of the diterpenoid compound in the preparation of drugs that reduce the expression of NLRP3 inflammasome, IL-1β and / or IL-18 in cells.

[0013] Furthermore, the cells mentioned are the human mononuclear cell line THP-1.

[0014] Furthermore, the cells mentioned are LPS-induced human mononuclear cell line THP-1 derived macrophages.

[0015] Furthermore, in the aforementioned applications, the concentration of the diterpenoid compound is 6-14 μM. Beneficial effects

[0016] This invention isolates a novel compound from *Callicarpa nudiflora*, which has been identified as a diterpenoid. Cellular experiments demonstrate that this compound significantly inhibits LPS-induced expression of the NLRP3 inflammasome in human monocyte THP-1 macrophages, and also significantly inhibits the expression of IL-1β and IL-18. This indicates that the compound possesses significant anti-inflammatory efficacy and can be used to inhibit inflammatory responses related to NLRP3 inflammasome activation and the production of cytokines IL-1β and IL-18. This provides a candidate compound for the development of anti-inflammatory drugs, and has significant potential application value, particularly in the treatment of inflammatory diseases of the skin, such as those involving wound healing, and in diseases or pathological processes accompanied by inflammation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 HR-ESI-MS spectrum of compound 1.

[0019] Figure 2 Compound 1 1 H-NMR (600 MHz, CDCl3).

[0020] Figure 3 Compound 1 13 C-NMR (125 MHz, CDCl3).

[0021] Figure 4 Calculation and experimental ECD spectra of compound 1.

[0022] Figure 5 The structural formula of compound 1.

[0023] Figure 6 Effects of compound 1 on LPS-induced expression of NLRP3 inflammasome, IL-1β, and IL-18 in human monocyte cell line THP-1 macrophages. Compared with the blank control group, * p < 0.05, ** p < 0.005, *** p < 0.001; compared with the model control group, # p < 0.05,## p < 0.01, ### p < 0.001. Where A represents the Western blotting result; BD represents the relative protein expression levels of NLRP3 (B), IL-1β (C), and IL-18 (D) in cells treated with compound 1, analyzed by Western blotting. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0025] Example 1: Preparation of the compound Take 36 kg of leaves of Callicarpa nudiflora and extract them by reflux at a volume concentration of 95%. After recovering the ethanol from the extract, dissolve the extract in water, extract with dichloromethane, recover the lower organic phase, concentrate under reduced pressure, and obtain dichloromethane extract (1500 g).

[0026] The obtained dichloromethane extract was subjected to silica gel column chromatography (silica gel 200-300 mesh, particle size 200-300 μM) with gradient elution using a mixed solution of petroleum ether and ethyl acetate. The volume ratios of the petroleum ether and ethyl acetate mixed solution were 100:0, 100:1, 100:2, 100:4, 100:5, 100:8, 100:10, 100:12, 100:20, 100:50, and 0:100.

[0027] A fraction obtained by mixing petroleum ether and ethyl acetate at a volume ratio of 100:10 was loaded onto a silica gel column (200-300 mesh silica gel, particle size 200-300 μM). Gradient elution was performed using a mixture of petroleum ether and ethyl acetate as the eluent, with the volume ratios of the petroleum ether and ethyl acetate mixtures being 40:1, 30:1, 25:1, 20:1, 15:1, 12:1, 10:1, 8:1, 5:1, 1:1, and 0:1, respectively. The fraction eluted at a petroleum ether and ethyl acetate volume ratio of 10:1 was further separated by semi-preparative high-performance liquid chromatography (HPLC) using methanol-0.2% formic acid water at a ratio of 52:48 to obtain compound 1. The high performance liquid chromatography (HPLC) conditions were as follows: column: YMC-pack ODS-A column (250 mm × 10 mm, 5 μm, 12 nm, YMC Co., Ltd., Kyoto, Japan); eluent: methanol-0.2% formic acid water = 73:27; flow rate: 2 mL / min; and retention time of compound 1: 58 min.

[0028] The HR-ESI-MS spectrum of compound 1 described in this invention is as follows: Figure 1 As shown, the nuclear magnetic resonance spectrum is as follows: Figures 2 to 3 Calculation and experimental ECD spectra, such as Figure 4 As shown. Spectral data are shown in Tables 1 and 2. The structural formula of compound 1 was identified based on the combined spectral data as follows. Figure 5 As shown.

[0029] Table 1. Spectral data of compound 1 of the present invention 1 1.76 (1H, m,Ha); 1.62 (1H, m, Hb) 32.3 2 2.51 (1H, m, Ha); 2.42 (1H, m, Hb) 27.8 3 — 175.4 4 — 146.7 5 2.26 (1H, m) 50.6 6 1.72 (1H, m, Ha); 1.65 (1H, m, Hb) 29.7 7 2.43 (1H, m, Ha); 2.04 (1H, m, Hb) 37.6 8 — 147.6 9 2.39 (1H, m) 44.0 10 — 41.5 11 1.77 (1H, m, Ha); 1.61 (1H, m, Hb) 31.7 12 4.61 (1H, m) 67.1 13 — 173.6 14 6.02 (1H, s) 114.4 15 — 173.7 16 4.94 (2H, brs) 71.1 17 4.96 (1H, brs, Ha); 4.45 (1H, brs, Hb) 107.2 18 4.89 (1H, brs, Ha); 4.72 (1H, brs, Hb) 114.0 19 1.75 (3H, s) 23.3 20 0.73 (3H, s) 17.6 21 4.15 (2H, q, = 7.5 Hz) 60.9 22 1.28 (3H, t, = 7.5 Hz) 14.2 Table 2 High-resolution mass spectrometry data of compound 1 Compound 1 HR-ESI-MS <![CDATA[[M+Na] + ]]> 399.2147 399.2147 <![CDATA[C 22 H 32 O5]]>

[0030] Example 2: Experiment on the inhibition of NLRP3 inflammasome activity by compound 1 of the present invention.

[0031] Methods: (1) Cell culture: The human mononuclear cell line THP-1 was cultured in RPMI-1640 medium containing 10% FBS, 50 μM mercaptoethanol and 1% penicillin / streptomycin solution, and maintained in a culture environment of 37°C and 5% CO2. The cell density was 5 × 10⁶ cells / year. 5 THP-1 monocytes were seeded at 100 ng / mL in six-well plates and incubated with 100 ng / mL PMA for 48 h to induce differentiation of THP-1 monocytes into adherent macrophages. After stabilization, subsequent experiments could be performed.

[0032] (2) Western blot analysis of the effects of diterpenoid compound 1 on the secretion of interleukin-1β (IL-1β), interleukin-18 (IL-18), and NLRP3 inflammasomes by LPS-activated human monocyte cell line THP-1: THP-1 cells were cultured in PMA-free medium for 24 h, then LPS (0.5 μg / mL) and different concentrations of compound 1 were added, and the cells were cultured for another 24 h. The cell supernatant was collected. Total protein was extracted using RIPA lysis buffer, and the protein concentration was detected by BCA method. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 h, incubated overnight at 4°C with primary antibody, and then incubated with HRP-labeled secondary antibody for 1 h. The gels were then visualized using a gel imaging system and an ultrasensitive chemiluminescence (ECL) detection kit.

[0033] Experimental results are as follows Figure 6 As shown: Compared with the blank control group, the expression of NLRP3 inflammasome, IL-1β, and IL-18 in the model control group was significantly increased, indicating successful modeling. The diterpenoid compound 1 described in this invention, at low (L) 6 μM and high (H) 14 μM treatment concentrations, was able to inhibit the secretion of NLRP3 inflammasome, IL-1β, and IL-18 by LPS-induced human monocyte cell line THP-1 macrophages. The relative expression levels of NLRP3 inflammasome, IL-1β, and IL-18 were all significantly reduced, showing significant differences compared to the model group. This invention demonstrates that the diterpenoid compound 1 significantly inhibits the expression of NLRP3 inflammasome, IL-1β, and IL-18 in LPS-induced human monocyte cell line THP-1 macrophages at treatment concentrations of 6 μM and 14 μM, exhibiting good anti-inflammatory activity. It can inhibit the inflammatory response mediated by NLRP3 inflammasome activation and the production of downstream cytokines IL-1β and IL-18, and can be used to treat or prevent inflammatory diseases mediated by NLRP3 inflammasome activation and the production of cytokines IL-1β and IL-18, especially in the treatment of skin wound inflammation associated with excessive activation of NLRP3 inflammasome, such as promoting skin wound healing. Compound 1 of this invention has significant application value.

[0034] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A diterpenoid compound, characterized in that, Its structural formula is shown in Formula I: 。 2. The method for extracting diterpenoid compounds as described in claim 1, characterized in that, Includes the following steps: (1) Take the dried leaves of Callicarpa nudiflora, heat and reflux them with 95% ethanol, recover the ethanol, dissolve them in water, and extract with dichloromethane to obtain dichloromethane extract. (2) The dichloromethane extract was separated by silica gel column chromatography and the eluent was obtained by gradient elution with a mixture of petroleum ether and ethyl acetate. (3) The fraction obtained by the volume ratio of petroleum ether and ethyl acetate of 100:10 was separated by silica gel column chromatography and eluted by gradient with a mixed solution of petroleum ether and ethyl acetate. The fraction eluted by the volume ratio of petroleum ether and ethyl acetate of 10:1 was further separated and purified by semi-preparative high performance liquid chromatography to obtain compound I.

3. The extraction method as described in claim 2, characterized in that, In steps (2) and (3), the silica gel used in the silica gel column is 200-300 mesh; in step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 100:0-0:100; in step (3), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 40:1-0:100, and the chromatographic column of the semi-preparative high performance liquid chromatography is a YMC-pack ODS-A column with a column length of 250 mm, an inner diameter of 10 mm, a particle size of 5 μm, and a micropore size of 12 nm.

4. The extraction method as described in claim 3, characterized in that, In step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 100:0, 100:1, 100:2, 100:4, 100:5, 100:8, 100:10, 100:12, 100:20, 100:50 and 0:100 respectively; in step (3), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 40:1, 30:1, 25:1, 20:1, 15:1, 12:1, 10:1, 8:1, 5:1, 1:1 and 0:1 respectively; in step (3), during the semi-preparative high performance liquid chromatography separation process, the eluent is methanol-0.2% formic acid water = 73:27, the flow rate is 2 mL / min, and the retention time of the compound of formula I is 58 minutes.

5. The use of the diterpenoid compound as described in claim 1 in the preparation of anti-inflammatory drugs.

6. The use of the diterpenoid compound as described in claim 1 in the preparation of a medicament for promoting wound healing.

7. The use of the diterpenoid compound as described in claim 1 in the preparation of a drug that reduces the expression levels of NLRP3 inflammasome, IL-1β and / or IL-18 in cells.

8. The application as described in claim 7, characterized in that, The cells described are the human mononuclear cell line THP-1.

9. The application as described in claim 7 or 8, characterized in that, The cells described are LPS-induced human mononuclear cell line THP-1 derived macrophages.

10. The application as described in claim 7 or 8, characterized in that, The concentration of the diterpenoid compound is 6-14 μM.

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