Knoevenagel condensation, preparation method and application thereof

Abstract

Knoxia vallicola terpenoid C, preparation method and application thereof, relate to a new substance with in-vitro anti-inflammatory activity and a method and application thereof. The present application provides a new compound with in-vitro anti-inflammatory activity and a preparation method and application thereof. Knoxia vallicola terpenoid C is chemically named as 8,14-dihydroxy-17,18,19,20-tetramethyl-decahydrophenanthro-furan-16-one, and has a molecular formula of C 20 H 28 O4. The preparation method comprises the following steps: 1, Knoxia vallicola is extracted by cold soaking with ethanol; 2, the extract of the recovered ethanol is dispersed with water, and then subjected to D101 macroporous resin; 3, the eluate of the macroporous resin subjected to 60% ethanol is repeatedly separated by a silica gel column chromatography, and then subjected to high performance liquid chromatography preparation, so that a monomer compound is obtained, which is identified as a new compound by spectroscopy and named as Knoxia vallicola terpenoid C. Knoxia vallicola terpenoid C is applied to preparation of anti-inflammatory drugs. The inhibition rates of 12.5 μM, 25.0 μM and 50.0 μM Knoxia vallicola terpenoid C on the release of pro-inflammatory factor TNF-α of LPS-induced mouse macrophage RAW264.7 are 9.2%, 21.2% and 40.7% respectively, and the inhibition rates on NO are 2.7%, 10.1% and 11.5% respectively, indicating that the compound has good in-vitro anti-inflammatory activity. The present application belongs to the field of extraction of natural medicine effective components.

Description

Technical Field

[0001] This invention relates to a novel substance with in vitro anti-inflammatory properties, its method, and its application. Background Technology

[0002] Euphorbia lunulata, listed in the 2025 edition of the Chinese Pharmacopoeia, is a traditional Chinese medicinal herb. It is bitter and pungent in taste, neutral in nature, and toxic. It has the effects of draining water and promoting urination, breaking up accumulations and killing parasites. It is mainly used to treat edema and abdominal distension, phlegm and food stagnation, abdominal pain, tuberculosis, scabies, etc.

[0003] Modern pharmacological studies have shown that the main active components of Euphorbia lunulata are diterpenoids, which have anti-tumor, anti-inflammatory, antibacterial, antifungal, antiviral and anti-epileptic effects, making it a valuable source of plant-based anti-inflammatory drugs.

[0004] Currently, there are many preparations of Euphorbia lunulae on the market. However, as a medicinal plant that has been used for a long time, it is of great significance to carry out systematic chemical separation of Euphorbia lunulae in order to improve its quality standards and discover more indicative components with anti-inflammatory activity.

[0005] Inflammation is a conservative, protective physiological response triggered by harmful signals such as pathogen infection, tissue damage, or toxic stimuli. This process coordinates the recruitment and activation of immune cells and the release of various inflammatory mediators to eliminate harmful stimuli, clear damaged tissue, and initiate tissue repair. However, uncontrolled or persistent inflammatory responses can disrupt tissue homeostasis, thereby inducing various inflammation-related pathological injuries. Therefore, substances that can inhibit inflammatory responses are of great significance for safeguarding public health and improving people's quality of life. Summary of the Invention

[0006] The purpose of this invention is to provide a novel compound with in vitro anti-inflammatory activity, its preparation method, and its application.

[0007] Euphorbia lunata C, wherein the molecular formula of Euphorbia lunata C is C 20 H 28 O4, the molecular structural formula is:

[0008] The molecular weight is 381.2195.

[0009] The preparation method of the euphorbia lycopersicate C is carried out according to the following steps:

[0010] 1. Place Euphorbia fischeriana in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with ethanol to obtain ethanol extract;

[0011] 2. After concentrating the extract obtained in step 1, disperse it with water onto D101 macroporous resin, and elute it sequentially with a mixed solvent of methanol and water at volume concentrations of 0%, 20%, 40%, and 60%, respectively.

[0012] 3. The eluent obtained in step 2, a mixture of methanol and water with a volume concentration of 60%, was separated by silica gel column chromatography. The eluent was then eluted sequentially with mixed solvents of petroleum ether and ethyl acetate at volume ratios of 100:0, 80:1, 50:1, and 30:1. The eluent with a petroleum ether to ethyl acetate volume ratio of 30:1 was further separated by silica gel column chromatography with dichloromethane as the eluting agent. The dichloromethane eluent was collected, and a high-performance liquid chromatography (HPLC) chromatogram was prepared using a mixed solvent of acetonitrile and water at a volume ratio of 65:35 as the mobile phase and a flow rate of 3 mL / min. The absorption peak appearing at the elution time of 34.0 min was collected, yielding Euphorbia lathyris C.

[0013] Furthermore, the pressure of the Soxhlet dynamic extraction and concentration unit described in step one is set to normal temperature and normal pressure extraction.

[0014] Furthermore, the high-performance liquid chromatography (HPLC) chromatograms described in step three were acquired using a Waters 2535 semi-preparative HPLC system, with C0.05... 18 The reversed-phase silica gel column was used as the packing material, and the Waters 2414 differential detector was used as the detector.

[0015] Furthermore, the application of the euphorbia lunata C, wherein the euphorbia lunata C is used as an active ingredient in the preparation of anti-inflammatory drugs.

[0016] Furthermore, the dosage forms of the anti-inflammatory drugs include injections, lyophilized powder injections, and oral preparations.

[0017] Furthermore, the oral formulations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release formulations.

[0018] Furthermore, the euphorbia pekinensis C, as the active ingredient of an anti-inflammatory drug, is used to inhibit LPS-induced TNF-α release from macrophages.

[0019] The chemical name of the euphorbia pekinenone of this invention is 7-hydroxyhugorosenone, and its molecular formula is C2. 20 H 30 O3.

[0020] The compounds described in this invention can be formulated into pharmaceutically acceptable solid or liquid formulations by adding pharmaceutically acceptable excipients.

[0021] There are no restrictions on the excipients used in this invention, as long as they are pharmaceutically acceptable.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention provides a novel compound derived from Euphorbia lunata and a method for extracting, separating, and purifying the novel compound. The method employs ethanol extraction, macroporous resin, silica gel column chromatography, and preparative high-performance liquid chromatography for separation and purification, successfully obtaining the novel compound. The operation method is simple and rapid, and the compound obtained by this method has high purity. Furthermore, studies have shown that the above compound has anti-inflammatory activity.

[0024] This novel compound can inhibit the increase in TNF-α and NO levels induced by LPS in mouse RAW264.7 macrophages. At concentrations of 12.5 μM, 25.0 μM, and 50.0 μM, the compound *Euphorbia lyratae* C exhibited inhibition rates of 9.2%, 21.2%, and 40.7% on TNF-α production, and 2.7%, 10.1%, and 11.5% on NO production, respectively. This indicates that it possesses good in vitro anti-inflammatory activity. *Euphorbia lyratae* C of this invention can be formulated into anti-inflammatory drugs such as injections, lyophilized powder for injection, infusions, or oral preparations (including tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release formulations). Attached Figure Description

[0025] Figure 1 This is the MS image of Euphorbia lathyris C;

[0026] Figure 2 This is the UV spectrum of Euphorbia lathyris C;

[0027] Figure 3 It is Euphorbia lathyris C. 1 H-NMR spectrum;

[0028] Figure 4 It is Euphorbia lathyris C. 13 C-NMR spectrum;

[0029] Figure 5 This is the DEPT spectrum of Euphorbia lathyris C;

[0030] Figure 6 This is the HSQC spectrum of Euphorbia lathyris C;

[0031] Figure 7 This is the HMBC spectrum of Euphorbia lathyris C;

[0032] Figure 8 It is Euphorbia lathyris C. 1 H- 1 H COSY spectrum;

[0033] Figure 9 This is the NOESY spectrum of Euphorbia lathyris C. Detailed Implementation

[0034] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0035] Specific Implementation Method 1: This implementation method uses Euphorbia lunulae C, wherein the molecular formula of Euphorbia lunulae C is C 20 H 28 O4, the molecular structural formula is:

[0036] The molecular weight is 381.2195.

[0037] Specific Implementation Method Two: The preparation method of Euphorbia lathyris C described in Specific Implementation Method One is carried out according to the following steps:

[0038] 1. Place Euphorbia fischeriana in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with ethanol to obtain ethanol extract;

[0039] 2. After concentrating the extract obtained in step 1, disperse it with water onto D101 macroporous resin, and elute it sequentially with a mixed solvent of methanol and water at volume concentrations of 0%, 20%, 40%, and 60%, respectively.

[0040] 3. The eluent obtained in step 2, a mixture of methanol and water with a volume concentration of 60%, was separated by silica gel column chromatography. The eluent was then eluted sequentially with mixed solvents of petroleum ether and ethyl acetate at volume ratios of 100:0, 80:1, 50:1, and 30:1. The eluent with a petroleum ether to ethyl acetate volume ratio of 30:1 was further separated by silica gel column chromatography with dichloromethane as the eluting agent. The dichloromethane eluent was collected, and a high-performance liquid chromatography (HPLC) chromatogram was prepared using a mixed solvent of acetonitrile and water at a volume ratio of 65:35 as the mobile phase and a flow rate of 3 mL / min. The absorption peak appearing at the elution time of 34.0 min was collected, yielding Euphorbia lathyris C.

[0041] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that the pressure of the Soxhlet dynamic extraction and concentration unit described in step 1 is set to ambient temperature and atmospheric pressure extraction. Everything else is the same as in Specific Implementation Method 2.

[0042] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods Two or Three in that the high-performance liquid chromatography (HPLC) chromatograms described in step three are acquired using a Waters 2535 semi-preparative HPLC system, with C0.05 as the standard value. 18 The reversed-phase silica gel column is used as the packing material, and a Waters 2414 differential detector is used as the detector. Everything else is the same as in specific embodiments two or three.

[0043] Specific Implementation Method 5: Application of Euphorbia lunata C described in Specific Implementation Methods 1 to 4, wherein Euphorbia lunata C is used as an active ingredient in the preparation of anti-inflammatory drugs.

[0044] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Five in that the dosage form of the anti-inflammatory drug includes injections, lyophilized powder injections, and oral preparations. Everything else is the same as in Specific Implementation Method Five.

[0045] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Six in that the oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations. Everything else is the same as in Specific Implementation Method Six.

[0046] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method Five in that the euphorbia pekinensis C is used as the active ingredient of an anti-inflammatory drug to inhibit LPS-induced TNF-α release from macrophages. Everything else is the same as in Specific Implementation Method Five.

[0047] The following experiments were used to verify the effectiveness of the invention:

[0048] Experiment 1:

[0049] Euphorbia lunata C, wherein the molecular formula of Euphorbia lunata C is C 20 H 28 O4, the molecular structural formula is:

[0050] The molecular weight is 381.2195.

[0051] The preparation method of the euphorbia lycopersicate C is carried out according to the following steps:

[0052] 1. Place Euphorbia fischeriana in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with ethanol to obtain ethanol extract;

[0053] 2. After concentrating the extract obtained in step 1, disperse it with water onto D101 macroporous resin, and elute it sequentially with a mixed solvent of methanol and water at volume concentrations of 0%, 20%, 40%, and 60%, respectively.

[0054] 3. The eluent obtained in step 2, a mixture of methanol and water with a volume concentration of 60%, was separated by silica gel column chromatography. The eluent was then eluted sequentially with mixed solvents of petroleum ether and ethyl acetate at volume ratios of 100:0, 80:1, 50:1, and 30:1. The eluent with a petroleum ether to ethyl acetate volume ratio of 30:1 was further separated by silica gel column chromatography with dichloromethane as the eluting agent. The dichloromethane eluent was collected, and a high-performance liquid chromatography (HPLC) chromatogram was prepared using a mixed solvent of acetonitrile and water at a volume ratio of 65:35 as the mobile phase and a flow rate of 3 mL / min. The absorption peak appearing at the elution time of 34.0 min was collected, yielding Euphorbia lathyris C.

[0055] The pressure of the Soxhlet dynamic extraction and concentration unit described in step one is set to normal temperature and normal pressure extraction.

[0056] The high-performance liquid chromatography (HPLC) chromatograms described in step three were acquired using a Waters 2535 semi-preparative HPLC system, with C0.05 as the chromatogram. 18 The reversed-phase silica gel column was used as the packing material, and the Waters 2414 differential detector was used as the detector.

[0057] This experiment used 30 kg of dried Euphorbia lunata, and finally obtained 1.60 mg of Euphorbia lunata terpene C. Table 1 shows the NMR data of the new compound Euphorbia lunata terpene C (using CD3OD). 1 H-NMR and 13 C-NMR test).

[0058] Table 1: NMR data of the novel compound Euphorbia lunata C of this invention

[0059]

[0060] The final molecular structure of Euphorbia lathyris C was determined to be:

[0061] .

[0062] Based on its structure, the euphorbia C compound was named 7-hydroxyhugorosenone.

[0063] Experiment 2:

[0064] The inhibitory activity of Euphorbia lathyris C prepared in Experiment 1 against TNF-α and NO production was investigated, and the steps were as follows:

[0065] 1. Drug preparation

[0066] Euphorbia pekinensis C was dissolved in DMSO to prepare a 500 μM stock solution. The solution was then diluted with PBS to test concentrations of 12.5 μM, 25.0 μM, and 50.0 μM.

[0067] 2. Cell Culture

[0068] Mouse macrophages RAW264.7 were grown in DMEM medium containing 10% fetal bovine serum and cultured in suspension in an incubator with 5% CO2, saturated humidity and 37°C. Cells in the logarithmic growth phase were used for experiments.

[0069] 3. Effects of CCK-8 assay on cell proliferation

[0070] RAW264.7 cells were grown at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates. After adhesion, the plates were treated with different concentrations of euphorbia lysine B and dexamethasone (positive control) for 24 h. The plates were then removed, the supernatant discarded, and 10 μL of CCK-8 solution and 90 μL of complete culture medium were added to each well. The plates were incubated for 45 min, and the absorbance (OD value) at 450 nm was measured using a microplate reader. Wells containing only CCK-8 solution and complete culture medium were designated as the complete blank group. All groups had at least three replicates. This was used to determine the safe concentration of euphorbia lysine C. The results showed that euphorbia lysine C had no cell-killing ability at the tested concentration.

[0071] Table 2 Effects of LPS and Euphorbia lysine C on cell viability

[0072]

[0073] 4. Inhibitory activity against TNF-α production

[0074] RAW 264.7 cells were grown at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / mL in 96-well plates. After adhesion, the cells were pretreated with different concentrations of Euphorbia lathyris C and dexamethasone (positive control) for 2 h, followed by incubation with 100 ng / mL LPS for 24 h. The required number of pre-coated strips were then removed and placed in 96-well frames. 100 μL of the cell culture supernatant was added to the corresponding wells. The wells were sealed with sealing film and incubated at room temperature for 120 min. The plates were washed 5 times, with the last wash performed by patting dry on thick absorbent paper. 100 μL of biotinylated antibody was added to each well. The wells were sealed with clear sealing film and incubated at room temperature for 60 min. The plates were washed 5 times, with the last wash performed by patting dry on thick absorbent paper. 100 μL of horseradish peroxidase-labeled Streptavidin was added to each well. The wells were sealed with white sealing film and incubated at room temperature in the dark for 20 min. The plates were washed 5 times, with the last wash performed by patting dry on thick absorbent paper. Add 100 μL of TMB chromogenic reagent per well, seal the wells with white sealing film, and incubate at room temperature in the dark for 20 min. Add 50 μL of stop solution (2 mol / L H2SO4 solution) per well, mix well, and immediately measure the A450 value. Repeat the above experiment three times consecutively. The results showed that the inhibition rates at concentrations of 12.5, 25.0, and 50.0 μM were 9.2%, 21.2%, and 40.7%, respectively, indicating that it has good in vitro anti-inflammatory activity.

[0075] 5. Inhibitory activity against NO production

[0076] RAW 264.7 cells were cultured at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / mL in 96-well plates. After cell adhesion, the cells were pre-treated for 2 h with different concentrations of Euphorbia lathyris B and the positive control drug dexamethasone, followed by incubation with 100 ng / mL LPS for 24 h. After incubation, the cell culture supernatant was collected. Samples containing precipitate were centrifuged, and the supernatant was used as the test sample. Before the experiment, Griess Reagent I and II were brought to room temperature. NO standard was diluted with DMEM + 10% FBS cell culture medium to prepare a 50 μM standard working solution. 50 μL of the standard solution and the test sample were added to the corresponding reaction wells in the 96-well plates. 50 μL of room temperature Griess Reagent I and 50 μL of room temperature Griess Reagent II were added to each well, and the mixture was thoroughly mixed to construct the colorimetric reaction system. Blank and standard groups were also set up. After the reaction, the absorbance of each well was measured at 540 nm immediately. The experiment was repeated three times, and the results showed that 12.5 μM, 25.0 μM, and 50 μM Euphorbia pekinensis C inhibited LPS-induced NO release from RAW264.7 cells by 2.7%, 10.1%, and 11.5%, respectively. This confirms that the compound can inhibit NO release from inflammatory cells and has good in vitro anti-inflammatory activity.

Claims

1. Euphorbia lathyris C, characterized in that, The molecular formula of the euphorbia lunulae C is C 20 H 28 O4, the molecular structural formula is: The molecular weight is 381.2195.

2. The method for preparing Euphorbia lathyris C according to claim 1, characterized in that, The preparation method of the euphorbia lycopersicate C is carried out according to the following steps:

1. Place Euphorbia fischeriana in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with ethanol to obtain ethanol extract; 2. After concentrating the extract obtained in step 1, disperse it with water onto D101 macroporous resin, and elute it sequentially with a mixed solvent of methanol and water at volume concentrations of 0%, 20%, 40%, and 60%, respectively.

3. The eluent obtained in step 2, a mixture of methanol and water with a volume concentration of 60%, was separated by silica gel column chromatography. The eluent was then eluted sequentially with mixed solvents of petroleum ether and ethyl acetate at volume ratios of 100:0, 80:1, 50:1, and 30:

1. The eluent with a petroleum ether to ethyl acetate volume ratio of 30:1 was further separated by silica gel column chromatography with dichloromethane as the eluting agent. The dichloromethane eluent was collected, and a high-performance liquid chromatography (HPLC) chromatogram was prepared using a mixed solvent of acetonitrile and water at a volume ratio of 65:35 as the mobile phase and a flow rate of 3 mL / min. The absorption peak appearing at the elution time of 34.0 min was collected, yielding Euphorbia lathyris C.

3. The method for preparing Euphorbia lathyris C according to claim 2, characterized in that, The pressure of the Soxhlet dynamic extraction and concentration unit described in step one is set to normal temperature and normal pressure extraction.

4. The method for preparing Euphorbia lathyris C according to claim 2, characterized in that, The high-performance liquid chromatography (HPLC) chromatograms described in step three were acquired using a Waters 2535 semi-preparative HPLC system, with C0.05 as the chromatogram. 18 The reversed-phase silica gel column was used as the packing material, and the Waters 2414 differential detector was used as the detector.

5. The application of the euphorbia pekinensis C according to claim 1, characterized in that, Euphorbia pekinensis C is used as an active ingredient in the preparation of anti-inflammatory drugs.

6. The application of Euphorbia lathyris C according to claim 5, characterized in that, The dosage forms of the anti-inflammatory drugs include injections, lyophilized powder injections, and oral preparations.

7. The application of Euphorbia lathyris C according to claim 6, characterized in that, The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

8. The application of Euphorbia lathyris C according to claim 5, characterized in that, Euphorbia pekinensis C, as the active ingredient of an anti-inflammatory drug, is used to inhibit LPS-induced TNF-α release from macrophages.

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