Application of sesquiterpenoids or medicinal derivatives thereof in preparation of medicines for preventing or treating inflammation

Abstract

The invention belongs to the field of natural products, and particularly relates to application of sesquiterpenoids or medicinal derivatives thereof in preparation of medicines for preventing or treating inflammation. Compared with the prior art, the invention has the following beneficial effects: the guaiane type sesquiterpene and uric acid dimer compound (the structural formula is as shown in formula I) extracted and separated from carpesium abrotanoides in Guizhou is found for the first time; the compound can significantly inhibit the content of inflammatory factors (such as TNF-alpha, IL-1alpha, IL-1beta, IL-6 and IL-18) of immune cells at extremely low concentrations (such as 20 nM, 10 nM, 5 nM and 1 nM), and can be used for preparing drugs for preventing or treating inflammation.

Description

Technical Field

[0001] This invention belongs to the field of natural products, and particularly relates to the use of a sesquiterpene compound or its pharmaceutical derivative in the preparation of a medicine for the prevention or treatment of inflammation. Background Technology

[0002] Inflammation is a core defense response of the body to injury or infection, but chronic inflammation can lead to serious consequences such as tissue fibrosis, autoimmune diseases, and malignant tumors, becoming a significant contributor to the global disease burden. Over the past two decades, anti-inflammatory drug development has evolved from traditional treatments such as nonsteroidal anti-inflammatory drugs (NSAIDs) to biologics and small-molecule inhibitors targeting cytokine signaling pathways (such as TNF-α and IL-6), forming a new paradigm of multi-target intervention. In this process, natural products, due to their advantages such as multi-component synergistic effects, extensive regulatory networks, and controllable side effects, have consistently been a key resource for anti-inflammatory drug discovery. Of the 89 anti-inflammatory drugs currently used clinically, small-molecule drugs account for approximately 65%, of which about 72% are directly derived from plant extracts or microbial secondary metabolites. Therefore, the discovery of anti-inflammatory active molecules based on natural products remains a crucial strategy for addressing inflammation-related diseases.

[0003] Tianming Jingshu ( Carpesium This plant is used in traditional Southeast Asian medicine for its anti-inflammatory and hemostatic properties. Its sesquiterpene lactones possess significant anti-inflammatory potential and are an important source for drug matrix modification. Furthermore, the plant contains other terpenes and secondary metabolites, making it valuable for anti-inflammatory drug development. Although some active ingredients of Guizhou Tianmingjing have been reported, its comprehensive chemical composition and anti-inflammatory mechanism still require systematic research.

[0004] This invention isolates guaiac sesquiterpenes and uric acid dimer compounds from Guizhou Tianmingjing, and preliminarily confirms their strong anti-inflammatory activity. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a dimeric compound of guaiac sesquiterpenes and uric acid, or its pharmaceutical derivatives, derived from Guizhou Tianmingjing, in the preparation of drugs for the prevention or treatment of inflammation. The compound provided by this invention has a significant inhibitory effect on inflammatory factors released by immune cells and can be used as an anti-inflammatory drug for the prevention or treatment of inflammatory diseases.

[0006] The technical solution of the present invention is as follows: This invention provides the use of a sesquiterpene compound or its pharmaceutical derivative in the preparation of a drug for the prevention or treatment of inflammation. The compound is a dimer of guaiacane-type sesquiterpenes and uric acid, and its structural formula is shown in Formula I. , Formula I.

[0007] Furthermore, in the aforementioned applications, the pharmaceutical derivatives of the compounds shown in Formula I can be pharmaceutically acceptable salts, esters, or stereoisomers of the compounds shown in Formula I.

[0008] Furthermore, in the aforementioned applications, the compound represented by Formula I or its pharmaceutical derivatives can be used alone or in combination with other drugs. The main purposes of combination therapy include enhancing therapeutic effects, reducing the occurrence of drug resistance, or reducing the dosage and side effects of a single drug.

[0009] Furthermore, in the aforementioned applications, the compound represented by Formula I or its pharmaceutical derivatives can be used in combination with a pharmaceutically acceptable carrier to form a drug formulation suitable for administration. Dosage forms of the drug formulation include, but are not limited to, tablets, capsules, granules, oral solutions, injections, patches, or inhalers. From the perspective of patient compliance and ease of administration, oral dosage forms are preferred.

[0010] Furthermore, in the aforementioned applications, the drug uses the compound shown in Formula I or its pharmaceutical derivatives as the active pharmaceutical ingredient, and the content of the active pharmaceutical ingredient is typically 0.1% to 99.9% of the total drug mass. The specific content can be designed according to the selected dosage form, route of administration, target dose, and course of treatment.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to discover that the dimeric compound of guaiac sesquiterpenes and uric acid extracted and isolated from Guizhou Tianmingjing (structural formula shown in Formula I) can significantly inhibit the content of inflammatory factors of immune cells (such as TNF-α, IL-1α, IL-1β, IL-6 and IL-18) at extremely low concentrations (such as 20 nM, 10 nM, 5 nM, 1 nM), and can be used to prepare drugs for the prevention or treatment of inflammation. Attached Figure Description

[0012] Figure 1 For compound 1 1 H NMR spectrum.

[0013] Figure 2 For compound 1 13 C10 NMR spectrum.

[0014] Figure 3 This is the HSQC spectrum of compound 1.

[0015] Figure 4 The image shows the HMBC spectrum of compound 1.

[0016] Figure 5 For compound 1 1 H- 1 H COSY spectrum.

[0017] Figure 6 The image shows the ROESY spectrum of compound 1.

[0018] Figure 7 The image shows the HR-ESI-MS spectrum of compound 1.

[0019] Figure 8 This is the IR spectrum of compound 1.

[0020] Figure 9 This is the UV spectrum of compound 1.

[0021] Figure 10 This is the CD spectrum of compound 1.

[0022] Figure 11 The effect of compound I and LPS (lipopolysaccharide) on THP-1 cell viability is shown. ns indicates no significant difference. The leftmost bar in the figure represents the control group without compound I and LPS.

[0023] Figure 12 This diagram shows the results of measuring the protein content of inflammatory factors secreted by immune cells using compound I of the present invention. Specifically, A represents the effect of compound I on the protein content of human tumor necrosis factor α (TNF-α) secreted by immune cells THP-1; B represents the effect of compound I on the protein content of human interleukin 1α (IL-1α) secreted by immune cells THP-1; C represents the effect of compound I on the protein content of human interleukin 1β (IL-1β) secreted by immune cells THP-1; and D represents the effect of compound I on the protein content of human interleukin 6 (IL-6) secreted by immune cells THP-1. Figure 12 The vertical axis represents the protein content of inflammatory factors secreted by immune cells, and the horizontal axis represents the treatment conditions. ns indicates no significant difference, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. The leftmost bar in the figure represents the control group without compound I and LPS.

[0024] Figure 13 The effect of compound I of the present invention on the RNA content of inflammatory factors in immune cells and its half-maximal inhibitory concentration (IC50) 50 The results are calculated as follows. A represents the effect of compound I on the RNA content of TNF-α in THP-1 immune cells; B represents the effect of compound I on the RNA content of IL-1β in THP-1 immune cells; C represents the effect of compound I on the RNA content of IL-6 in THP-1 immune cells; and D represents the effect of compound I on the RNA content of human interleukin-18 (IL-18) in THP-1 immune cells. Figure 13The vertical axis represents the RNA content of inflammatory factors in immune cells, and the horizontal axis represents the treatment conditions. ns indicates no significant difference, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. The leftmost bar in the figure represents the control group without compound I and LPS. Detailed Implementation

[0025] The sesquiterpene compound 1 isolated and purified from Guizhou Tianmingjing in this invention has the structure shown in Formula I: , Formula I.

[0026] The biological materials involved in the embodiments of this invention are described as follows: the human immune cells are the human immune cell line THP-1, purchased from the ATCC cell bank, catalog number TIB-202.

[0027] Unless otherwise specified, all reagents used in the embodiments of this invention can be purchased commercially. RPMI 1640 medium, LPS, and fetal bovine serum were purchased from Gibco. The ELISA kits used to detect immune factors in the cell culture medium were purchased from Shanghai Duma Biotechnology Co., Ltd., with catalog numbers DM4670 (human interleukin-6 ELISA kit), DM4677 (human tumor necrosis factor-α ELISA kit), DM4708 (human interleukin-1β ELISA kit), and DM4656 (human interleukin-1α (IL-1α) ELISA kit).

[0028] The cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2 and 90% humidity.

[0029] Example 1: Extraction and separation of guaiacane-type sesquiterpenes and uric acid dimer compound 1 (1) The aerial parts of *Guizhou Tianmingjing* were refluxed and extracted three times with methanol solution. The extracts were collected and concentrated under reduced pressure to obtain an extract. The extract was dispersed in an appropriate amount of water and extracted with petroleum ether and ethyl acetate respectively to obtain petroleum ether extract and ethyl acetate extract. The ethyl acetate extract was dissolved and mixed with 40-80 mesh silica gel. The sample was subjected to column chromatography with 100-200 mesh separating silica gel. The sample was eluted sequentially with petroleum ether-ethyl acetate and dichloromethane-methanol. Nine fractions of eluent were collected and named Fr. A to Fr. I respectively. The volume ratio of petroleum ether to ethyl acetate was 50:1 to 5:1. The volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture was 50:1 to 1:1. (2) Fr. H was separated by small-pore resin chromatography, and gradient elution was performed sequentially using methanol aqueous solution with a volume fraction of 50% to 100%. The eluents were collected and named Fr. Ga to Fr. Gf, respectively. Fr. Gd was subjected to C... 18 Reversed-phase silica gel column chromatography was used for separation, with gradient elution using methanol-water solutions of 50%–90% (v / v), collecting five eluent fractions, named Fr. Gd1–Fr. Gd5. Fr. Gd3 was eluted using Sephadex LH-20 methanol gel column chromatography, collecting 4 mL fractions of the chromatogram, collecting five eluent fractions, named Fr. Gd3a–Fr. Gd3e. The conditions for Sephadex LH-20 methanol gel column chromatography included: Eluent: Analytical grade methanol; Gel column dimensions: 15 mm * 160 mm; The flow rate of the eluent is 0.5 mL / min; The amount of elution solution used: 1.8 L; (3) Fr. Gd3d was separated by normal-phase silica gel column chromatography, eluted with dichloromethane-methanol, and three eluent fractions were collected and named Fr. Gd3d I to Fr. Gd3d III, with a volume ratio of dichloromethane to methanol of 20:1 to 8:1; Fr. Gd3d II was purified by preparative HPLC to obtain guaiacol-type sesquiterpenes and uric acid dimer compound 1. The preparative HPLC conditions included: Column specifications: 4.6 mm * 250 mm, 5 μm; Mobile phase: Acetonitrile-water 63:37; Flow rate: 2 mL / min.

[0030] The structural characterization of the guaiac sesquiterpene and uric acid dimer compound 1 is as follows: Figure 1-10 As shown in Table 1: Figure 1 Compound 1 is a guaiac sesquiterpene and uric acid dimer. 1 H NMR spectrum; Figure 2 Compound 1 is a guaiac sesquiterpene and uric acid dimer. 13 C NMR spectrum; Figure 3 The HSQC spectra of guaiac sesquiterpenes and uric acid dimer compound 1 are shown. Figure 4 HMBC spectra of guaiac sesquiterpenes and uric acid dimer compound 1; Figure 5 Compound 1 is a guaiac sesquiterpene and uric acid dimer. 1 H- 1 H COSY spectrum; Figure 6 The ROESY spectra of guaiac sesquiterpenes and uric acid dimer compound 1; Figure 7 HR-ESI-MS spectra of guaiac sesquiterpenes and uric acid dimer compound 1; Figure 8 The IR spectra of guaiac sesquiterpenes and uric acid dimer compound 1 are shown. Figure 9 The UV spectra of guaiac sesquiterpenes and uric acid dimer compound 1 are shown. Figure 10 The CD spectrum of compound 1 is shown for guaiac sesquiterpenes and uric acid dimers.

[0031] Compound 1 1 H-NMR and 13 The C-NMR data are shown in Table 1: Table 1. Guaiacane-type sesquiterpenes and uric acid dimers. 1 H-NMR and 13 C-NMR data

[0032] From Table 1 and Figures 1-10 The structural data of guaiac sesquiterpenes and uric acid dimer compound 1 are as follows: white amorphous powder.

[0033]

[0034] Infrared spectroscopy indicates the presence of hydroxyl groups (3435 cm⁻¹) in its structure. -1 ), ester carbonyl (1762 cm) -1 ) and olefin double bond (1626 cm) -1 ). 1 H and 13 C10 NMR data indicate that its structure contains two methyl groups. δ H 1.75 (3H, s), 0.97 (3H, s); δ C 20.8 (q), 23.6 (q)], 1 olefin double bond [ δ H 5.66 (1H, t, J = 1.8 Hz); δ C125.0(s), 141.4(t)] and 1 ester carbonyl group [ δ C [175.6 (s)] indicates that compound 1 contains a guaiacol-type sesquiterpene skeleton. Further analysis of the remaining NMR data... δ H 4.34, 10.7, 11.1, 11.7 δ C 97.6, 138.4, 149.5, 152.2, and 152.7 form a uric acid structure, which is related to HMBC H2-12 ( δ H 3.94, d, J = 7.8 Hz) to C-16 ( δ C 138.4) and C-17 ( δ C 149.5), indicating that the 12-position of the guaiacol-type sesquiterpene skeleton is replaced by uric acid, forming a guaiacol-type sesquiterpene and uric acid dimer. Simultaneously, through... 1 H- 1 The related signals from H COSY, HMBC, and ROESY were also confirmed to be guaiacol-type sesquiterpenes and uric acid dimers. Compound 1 was named Carpespene K.

[0035] The structural formula of compound 1 is shown in Formula I: , Formula I.

[0036] Example 2: Detection of the anti-inflammatory activity of compound I Compound I (i.e., compound 1 in Example 1) was dissolved in DMSO (dimethyl sulfoxide), and then solutions of each compound with concentrations of 20 nM, 10 nM, 5 nM and 1 nM were prepared using DMSO; the above concentration solutions were used as test solutions.

[0037] 1. To determine whether compound I and LPS (lipopolysaccharide) themselves cause cell damage and death, the CCK-8 assay kit was used to detect cell viability. The CCK-8 method was used to detect cell growth inhibition. The CCK-8 assay, short for Cell Counting Kit-8, is a commonly used method for detecting cell proliferation and cytotoxicity. The principle of the CCK-8 assay is based on the compound WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt), which, in the presence of the electron carrier 1-Methoxy PMS (menaquinone phosphate), can be reduced by intracellular mitochondrial dehydrogenases to generate a highly water-soluble orange-yellow formazan product. The amount of formazan generated is directly proportional to the number of viable cells, and the color intensity reflects cellular metabolic activity. The number of viable cells can be indirectly reflected by measuring the absorbance at 450 nm using a microplate reader. Therefore, CCK-8 can be used to assess cell proliferation, cytotoxicity, or the cellular effects of drugs. The specific steps for CCK-8 testing are as follows: When performing CCK-8 testing, use 3×10 5 THP-1 cells were seeded into five replicate wells of a 96-well plate at 90 µL of culture medium and cultured for 24 hours. Afterward, the cells were treated with different concentrations of Compound I and LPS. Forty-eight hours after treatment, 10 µL of CCK-8 reagent was added to each well, and the 96-well plate was incubated at 37 °C for 1 hour. The absorbance was measured at 450 nm using a microplate reader. The average OD value of each well was calculated and compared with the control group (without Compound I or LPS) to analyze changes in cell proliferation or viability. The percentage of absorbance value in the experimental group relative to the absorbance value in the control group represents the cell viability or proliferation level; the control group was assumed to be 100%.

[0038] The test results are as follows Figure 11 As shown, the addition of LPS and compound I (at concentrations of 20 nM, 10 nM, or 5 nM) did not inhibit the viability of THP-1 cells. This indicates that compound I is well-tolerated and does not inhibit the growth of immune cells. ns represents no statistically significant difference.

[0039] 2. To detect the anti-inflammatory activity of compound I, an ELISA kit was used to test whether compound I affected the levels of TNF-α, IL-1α, IL-1β, and IL-6 secreted by immune cells THP-1. The specific experimental procedure was as follows: THP-1 cells were treated with LPS for 24 h, then treated with compound I for 12 h. The cell suspension was collected, centrifuged, the precipitate was discarded, and the supernatant was collected. The levels of TNF-α, IL-1α, IL-1β, and IL-6 secreted by THP-1 cells were detected according to the ELISA kit instructions. Figure 12The vertical axis represents the expression level of inflammatory factors, and the horizontal axis represents the treatment conditions. ns indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and *** indicates p<0.0001.

[0040] TNF-α is a core driver and key regulator of inflammatory responses, primarily produced by activated immune cells. Through mechanisms such as activating vascular endothelial cells, recruiting and activating leukocytes, and inducing the release of other pro-inflammatory mediators, it powerfully initiates and amplifies local and systemic inflammatory responses, playing a crucial role in the body's resistance to infection and damage repair. However, excessive or persistent production can disrupt the balance between pro-inflammatory and anti-inflammatory responses, leading to tissue damage and becoming a core culprit in the pathological damage of various chronic inflammatory diseases (such as rheumatoid arthritis and inflammatory bowel disease). This also makes it a revolutionary target for anti-inflammatory therapy. IL-6 is a marker of early inflammation in inflammatory responses. IL-6 is associated with various autoimmune diseases; for example, serum IL-6 levels are typically high in patients with rheumatoid arthritis, psoriasis, and systemic lupus erythematosus. IL-1β plays an important role in the pathogenesis of acute and chronic inflammation and is closely related to the pathological processes of diabetes, rheumatoid arthritis, and periodontitis. IL-1α is a key pro-inflammatory cytokine, playing a pioneering and core driver role in the initiation and amplification of inflammatory responses. Sustained action of IL-1α can exacerbate inflammation and lead to pathological damage. IL-18 is a pleiotropic pro-inflammatory cytokine; its overactivation in chronic inflammation (such as rheumatoid arthritis and inflammatory bowel disease) or autoimmune diseases can lead to tissue damage. Therefore, TNF-α, IL-1α, IL-1β, IL-6, and IL-18 are important inflammatory targets.

[0041] The test results are as follows Figure 12 As shown, compound I significantly inhibited LPS-induced expression of inflammatory factors at extremely low concentrations (20 nM, 10 nM, 5 nM, and 1 nM treatments), and reduced the levels of TNF-α, IL-1α, IL-1β, and IL-6 secreted into the culture medium by LPS-induced THP-1 cells in a concentration-gradient manner. This demonstrates that compound I possesses extremely strong anti-inflammatory activity.

[0042] 3. To further detect the anti-inflammatory activity of compound I, IC50 was used. 50 The following experiment was conducted: THP-1 immune cells were treated with lipopolysaccharide (LPS) for 24 h, transforming them into inflammatory cells releasing inflammatory factors. Compound I was then added and treated for 12 h. RNA was extracted, and RT-qPCR was performed to detect the levels of 18S, TNF-α, IL-1β, IL-6, and IL-18. 18S was used as an internal control for statistical analysis. The RT-qPCR primer sequences are as follows:

[0043] Experimental results are as follows Figure 13 As shown.

[0044] Figure 13 A showed that LPS treatment of THP-1 cells induced an increase in the mRNA expression level of the inflammatory cytokine TNF-α. Compound I significantly reduced the LPS-induced mRNA content of the inflammatory cytokine TNF-α and inhibited the IC50 of TNF-α. 50 It is 6.81 nM.

[0045] Figure 13 B showed that LPS treatment of THP-1 cells induced an increase in the mRNA expression level of the inflammatory cytokine IL-1β, and compound I significantly reduced the LPS-induced mRNA content of the inflammatory cytokine IL-1β. Compound I inhibited the IC50 of IL-1β. 50 It is 9.53 nM.

[0046] Figure 13 C showed that LPS treatment of THP-1 cells induced an increase in the mRNA expression level of the inflammatory cytokine IL-6. Compound I significantly reduced the LPS-induced mRNA content of the inflammatory cytokine IL-6 and inhibited the IC50 of IL-6. 50 It is 4.75 nM.

[0047] Figure 13 D showed that LPS treatment of THP-1 cells induced an increase in the mRNA expression level of the inflammatory cytokine IL-18. Compound I significantly reduced the LPS-induced mRNA content of the inflammatory cytokine IL-18 and inhibited the IC50 of IL-18. 50 It is 9.36 nM.

[0048] In summary, compound I significantly reduced the expression levels of LPS-induced inflammatory factors TNF-α, IL-1α, IL-1β, IL-6, and IL-18 at low concentrations without affecting immune cell survival, demonstrating that compound I can inhibit the expression of inflammatory factors and has a strong anti-inflammatory effect.

[0049] In summary, compound I exhibits extremely strong anti-inflammatory effects, effectively inhibiting the release of inflammatory factors TNF-α, IL-1α, IL-1β, IL-6, and IL-18 even at very low concentrations.

[0050] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention extends to all other methods and applications having the same function.

Claims

1. Use of a sesquiterpene compound or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the prevention or treatment of inflammation, characterized in that, The compound is a dimer compound of guaiane sesquiterpene and uric acid, and its structural formula is shown as formula I. Formula I.

2. Use according to claim 1, characterized in that, The pharmaceutical derivative is a pharmaceutically acceptable salt, ester or stereoisomer of the compound shown as formula I.

3. Use according to claim 1, characterized in that, The compound shown as formula I or its pharmaceutical derivative is used alone or in combination with other drugs.

4. Use according to claim 1, characterized in that, The compound shown as formula I or its pharmaceutical derivative is used in combination with a pharmaceutically acceptable carrier to form a pharmaceutical preparation suitable for administration.

5. Use according to claim 4, characterized in that, The dosage form of the pharmaceutical preparation is tablet, capsule, granule, oral liquid, injection, patch or inhalation.

6. Use according to claim 1, characterized in that, The medicine uses the compound shown as formula I or its pharmaceutical derivative as the active pharmaceutical ingredient.

7. Use according to claim 6, characterized in that, In the medicine, the content of the active pharmaceutical ingredient is 0.1% to 99.9% of the total mass of the medicine.

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