Anti-inflammatory compound as well as preparation method and application thereof
By extracting and isolating anti-inflammatory compound 3 from pig shit bean seeds, the problem that pig shit bean pharmacological characteristics in the prior art are not fully utilized, and effective preparation of anti-inflammatory drugs is achieved, with significant anti-inflammatory effects and economic benefits.
Patent Information
- Application Number
- CN202510321647.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-20
AI Technical Summary
In the prior art, there are few scientific pharmacological characteristics of pig shit beans, and it is difficult to effectively use their anti-inflammatory properties to prepare drugs.
Anti-inflammatory compounds were extracted from pig shit bean seeds, and anti-inflammatory compounds 3 were prepared by 80% methanol reflux extraction, reverse phase chromatography separation and liquid chromatography purification, and pharmaceutical compositions were prepared using them as active ingredient.
The anti-inflammatory compound 3 was successfully isolated, which significantly inhibited the LPS-induced THP-1 macrophage inflammation model, had good anti-inflammatory activity, enriched the types of plant-based drugs, and the preparation method was simple and easy to implement.
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Figure CN120173033A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to an anti-inflammatory compound, a preparation method thereof, and uses thereof. Background Art
[0002] Crotalaria belongs to the legume family and is distributed in tropical and subtropical regions around the world. Plants of this genus are known as rattles or snake rattles by people because of the sound made when their pod-like fruits shake. In some countries such as Brazil and India, Crotalaria is used as a folk medicine for treating various diseases. The whole plant soaked can be used to treat skin infections and thrush. The roots can be used to treat hemoptysis, and the leaves and flowers can improve symptoms of fever, colds, and lung diseases. The powder of seeds boiled with milk can be used to treat skin diseases, leprosy, flatulence, and fever, and can also be used to relieve the pain of scorpion stings. Some articles have reported good pharmacological properties of Crotalaria plants, such as antioxidant, wound healing, anti-inflammatory, and antinociceptive activities.
[0003] Phytochemical studies on Crotalaria seeds have shown that it contains alkaloids, flavonoids, carbohydrates, free fatty acids, oils, minerals, and proteins. Although Crotalaria is widely used in folk medicine because of its pharmacological properties, there is little scientific information to confirm its properties.
[0004] Therefore, extracting effective pharmaceutical components from Crotalaria is of great significance for preparing drugs for preventing and / or treating inflammatory diseases. Summary of the Invention
[0005] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide an anti-inflammatory compound, a preparation method thereof, and uses thereof.
[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:
[0007] The present invention provides an anti-inflammatory compound or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, and the structure of the anti-inflammatory compound is shown in Formula I:
[0008]
[0009] Further, the pharmaceutically acceptable salt is selected from acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate, mesylate, sulfate, bisulfate, nitrate, oxalate, phosphate, or succinate.
[0010] The present invention also provides a preparation method of the above anti-inflammatory compound, and the preparation method includes the following steps:
[0011] (1) Extract Crotalaria seeds with an organic solvent;
[0012] (2) Concentrate, extract, and concentrate to obtain an extract.
[0013] (3) Elute the extract to obtain a crude extract.
[0014] (4) Separate the crude extract to obtain an anti-inflammatory compound.
[0015] Furthermore, in step (1), the organic solvent is 80% methanol, the volume ratio of the Crotalaria seeds to the organic solvent is 1:10, the extraction is reflux extraction; the extraction time is 2 h, and the number of extractions is 2 times.
[0016] Furthermore, in step (2), the extractants for extraction are petroleum ether, dichloromethane, ethyl acetate, and n-butanol in sequence, and the number of extractions is three times for each of petroleum ether, dichloromethane, ethyl acetate, and n-butanol.
[0017] Furthermore, in step (3), the elution is carried out through a reverse-phase chromatographic column, the eluents for elution are water, 20% methanol, 50% methanol, and methanol in sequence, the packing for elution is a reverse-phase polymer chromatographic packing, and the crude extract is the component eluted with 50% methanol collected.
[0018] Furthermore, in step (4), the separation is liquid chromatography separation, the mobile phase for separation consists of mobile phase A and mobile phase B, mobile phase A: acetonitrile, mobile phase B: 0.3% phosphoric acid aqueous solution, and the separation conditions are: 25% A from 0 - 40 min, 40% A at 60 min, and the detection wavelength is 320 nm.
[0019] The present invention also provides an anti-inflammatory pharmaceutical composition, which is prepared with the above anti-inflammatory compound or its pharmaceutically acceptable salt, stereoisomer, or solvate as the active ingredient, plus pharmaceutically acceptable excipients.
[0020] Furthermore, the pharmaceutical composition is a tablet, granule, or injection.
[0021] The present invention also provides the use of the above anti-inflammatory compound or its pharmaceutically acceptable salt, stereoisomer, or solvate in the preparation of a drug for preventing and / or treating inflammatory diseases.
[0022] The present invention has achieved the following beneficial effects:
[0023] The present invention has for the first time extracted and separated an anti-inflammatory compound - compound 3 from the natural medicinal material Crotalaria. The method is simple, easy to operate, safe, non-toxic, green, sustainable, and environmentally friendly.
[0024] The anti-inflammatory compound provided by the present invention can significantly inhibit the LPS-induced THP-1 macrophage inflammation model. It can be seen that the anti-inflammatory compound has good anti-inflammatory activity and can be used as an anti-inflammatory drug, enriching the types of plant extract drugs. The preparation method of the anti-inflammatory compound is simple and easy to implement, with good economic benefits and application prospects.
[0025] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.
[0026] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings
[0027] Figure 1 It shows the effects of compound 3 at different concentrations on the secretion level of IL-1β induced by LPS.
[0028] Figure 2 It shows the effects of compound 3 at different concentrations on the phosphorylation expression level of P65 induced by LPS. Detailed Description of the Embodiments
[0029] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.
[0030] The reverse-phase polymer chromatography packing used in the following Example 1 is the packing with the product model of Navi NM 200 purchased from Suzhou Navi Technology Co., Ltd.
[0031] In the following experiments, if the temperature is not specified, the reaction is carried out under normal temperature conditions. Normal temperature means room temperature, which is 25 ± 5°C.
[0032] Example 1: Preparation of Compound 3 of the Present Invention
[0033] Take 10 kg of Crotalaria pallida seeds, crush them, add 10 times the volume of 80% methanol, reflux and extract at 80 °C for 2 times, 2 hours each time. Recover the solvent under reduced pressure until the extract has no alcohol smell. Extract 3 times with petroleum ether, dichloromethane, ethyl acetate, and n-butanol respectively. Take the n-butanol layer, concentrate it until there is no solvent, and dissolve it in an appropriate amount of water. The extract is separated by passing through a reverse-phase chromatographic column (the packing is a reverse-phase polymer chromatographic packing: Navi NM 200), eluted successively with water, 20% methanol, 50% methanol, and methanol, 6 column volumes for each gradient elution, collected and concentrated under reduced pressure respectively, denoted as Fa, Fb, Fc, and Fd. Fb is separated by preparative liquid chromatography, and the conditions are: mobile phase A: acetonitrile, mobile phase B: 0.3% phosphoric acid aqueous solution, 15% A from 0 to 40 min, 60% A at 60 min, detection wavelength 320 nm. Fb-1 to Fb-7 are separated. Fc is separated by preparative liquid chromatography, and the conditions are: mobile phase A: acetonitrile, mobile phase B: 0.3% phosphoric acid aqueous solution, 25% A from 0 to 40 min, 40% A at 60 min, detection wavelength 320 nm. Fc-1 to Fc-8 are separated. Fc-3 is the compound 3, with a yield of 0.004% and a purity of 95%.
[0034] The hydrogen spectrum information of compound 3 is as follows:
[0035] 1H-NMR(600 MHz, CD3OD): 4.57 (1H, d, J = 5.2 Hz, H-1), 3.65 (1H, q, J = 5.3 Hz, H-2), 3.47–3.38 (1H, m, H-3α), 3.56–3.50 (1H, m, H-3β), 6.72 (2H, s, H-2', 6'), 4.80 (1H, d, J = 7.6 Hz, H-1”), 3.56–3.50 (1H, m, H-2”), 3.47–3.38 (1H, m, H-3”), 3.47–3.38 (1H, m, H-4”), 3.47–3.38 (1H, m, H-5”), 4.41 (1H, d, J = 11.8 Hz, H-6”α), 4.36–4.30 (1H, m, H-6”β), 7.19 (d, J = 2.2 Hz, H-2”'), 6.83 (1H, dd, J = 8.2, 1.3 Hz, H-5”'), 7.07 (1H, dd, J = 8.2, 2.2 Hz, H-6”'), 7.60 (1H, d, J = 15.9 Hz, H-7”'), 6.34 (1H, dd, J = 15.9, 1.4 Hz, H-8”'), 3.82 (6H, s, 3’-OCH3, 5’-OCH3), 3.90 (3H, s, 3”’-OCH3). 13C-NMR(151 MHz, CD3OD): 73.77 (C-1), 75.87 (C-2), 63.13 (C-3), 139.13 (C-1’), 104.23 (C-2’, 6’), 152.85 (C-3’, 6’), 133.95 (c-4’), 104.13 (C-1”), 74.24 (C-2”), 76.35 (C-3”), 70.26 (C-4”), 63.13 (C-6”), 126.32 (C-1”’), 113.95 (C-2”’), 149.30 (C-3”’), 145.57 (C-4”’), 115.17 (C-5”’), 122.79 (C-6”’), 148.04 (C-7”’), 110.39 (C-8”’), 167.56 (C-9”’) 55.57 (3’, 5’-OCH3), 55.16 (3”’-OCH3).
[0036] The beneficial effects of the present invention are demonstrated by the following experimental examples.
[0037] Experimental Example 1: Effect of Compound 3 of the Present Invention on the Secretion Level of IL-1β Induced by LPS
[0038] Take 1x10 6THP-1 cells were seeded in 6-well plates, with 2 mL of DMEM medium in each well. Different concentrations of Compound 3 (0 μg / mL, 300 μg / mL, 600 μg / mL) were mixed with LPS (1 μg / mL) and used to treat the cells simultaneously for 16 h. The cell culture suspension was centrifuged at 1500 rpm for 15 min at 4°C, and the supernatant was taken. The secreted IL-1β was detected using a commercially available ELISA kit (Abclonal, China) according to the instructions provided by the manufacturer. The remaining cells were washed 3 times with pre-cooled PBS, resuspended in 200 μL of RIPA lysis buffer (P0038, Beyotime), and lysed by sonication. After sufficient lysis, the mixture was centrifuged at 10,000 - 14,000×g for 3 - 5 min, and the supernatant was taken, which was the lysed supernatant for subsequent Western blot operations. The primary antibody p-P65 (ser536) was purchased from CST; β-actin was purchased from Zhongshan Jinqiao.
[0039] An LPS (concentration of 1 μg / mL)-induced THP-1 macrophage inflammation model was established to test the secretion levels of IL-1β in the cell culture supernatants of different groups. The results are as Figure 1 shown. The stimulation of LPS significantly increased the secretion of IL-1β in THP-1 macrophages, indicating that the THP-1 macrophage inflammation model was successfully established. Under the treatment of 300 μg / mL of Compound 3, Compound 3 had no significant effect on the secretion of the inflammatory factor IL-1β by LPS-induced THP-1 macrophages. Under the treatment of 600 μg / mL of Compound 3, Compound 3 could significantly inhibit the secretion of the inflammatory factor IL-1β by LPS-induced THP-1 macrophages.
[0040] Experimental Example 2: Effect of Compound 3 of the present invention on the phosphorylation expression level of P65 induced by LPS
[0041] The effect of different concentrations of Compound 3 on the phosphorylation level of P65 (a core molecule of the NF-κB pathway, belonging to one of the classical inflammatory pathways) in THP-1 macrophages stimulated by LPS was detected by Western blot.
[0042] The detection method is as follows:
[0043] The supernatant after lysis obtained in Experimental Example 1 was subjected to 10% SDS-polyacrylamide gel electrophoresis to separate proteins. Subsequently, the separated proteins were transferred to a PVDF membrane (Millipore) by electrotransfer. Before incubating with the primary antibody, the PVDF membrane containing the proteins was first blocked with a blocking solution (5% non-fat milk powder dissolved in TBST solution) at room temperature for 1 hour, and then washed 3 times with TBST buffer. To detect the phosphorylation of P65 in THP-1 macrophages, we used the Anti-p-P65 (ser536) primary antibody, diluted at a ratio of 1 / 1000 and incubated with the PVDF membrane at room temperature for 1 hour. Then it was incubated with the HRP secondary antibody for 1 hour, and then chemiluminescence color development was carried out.
[0044] The results are as Figure 2 shown. The stimulation of LPS can significantly up-regulate the level of P65 phosphorylation in THP-1 macrophages in vivo, and the treatment with Compound 3 can inhibit the up-regulation of P65 phosphorylation induced by LPS. Among them, Compound 3 has the best inhibitory effect at a concentration of 600 μg / mL.
[0045] In summary, the anti-inflammatory compound provided by the present invention can significantly inhibit the LPS-induced THP-1 macrophage inflammation model. It can be seen that the anti-inflammatory compound has good anti-inflammatory activity and can be used as an anti-inflammatory drug, enriching the types of plant-derived drugs. The preparation method of the anti-inflammatory compound is simple and feasible, with good economic benefits and application prospects.
Claims
1. An anti-inflammatory compound or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, characterized in that: The structure of the anti-inflammatory compound is shown in Formula I:
2. The anti-inflammatory compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, characterized in that: The pharmaceutically acceptable salt is selected from acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate, methanesulfonate, sulfate, bisulfate, nitrate, oxalate, phosphate or succinate.
3. The method for preparing the anti-inflammatory compound according to claim 1, characterized in that: The preparation method comprises the following steps: (1) extracting Crotalaria officinalis seeds with an organic solvent; (2) concentrating, extracting, and concentrating to obtain an extract; (3) eluting the extract to obtain a crude extract; (4) Separating the crude extract to obtain anti-inflammatory compounds.
4. The preparation method according to claim 3, characterized in that: The organic solvent in step (1) is 80% methanol, the volume ratio of the Crotalaria seeds to the organic solvent is 1:10, the extraction is reflux extraction; the extraction time is 2 hours, and the number of extractions is 2 times.
5. The preparation method according to claim 3, characterized in that: The extraction agents used in step (2) are petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence, and the extraction is performed three times with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively.
6. The preparation method according to claim 3, characterized in that: The elution in step (3) is elution through a reverse phase chromatography column, the eluents are water, 20% methanol, 50% methanol and methanol in sequence, the elution filler is a reverse phase polymer chromatography filler, and the crude extract is the component collected by 50% methanol elution.
7. The preparation method according to claim 3, characterized in that: The separation in step (4) is liquid chromatography separation, and the mobile phase for separation consists of mobile phase A and mobile phase B, mobile phase A: acetonitrile, mobile phase B: 0.3% phosphoric acid aqueous solution, and the separation conditions are: 0-40min 25% A, 60min 40% A.
8. An anti-inflammatory pharmaceutical composition, characterized in that: The pharmaceutical composition is prepared by using the anti-inflammatory compound or a pharmaceutically acceptable salt, stereoisomer or solvate thereof as an active ingredient and pharmaceutically acceptable excipients.
9. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition is in the form of tablets, granules or injections.
10. Use of the anti-inflammatory compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt, stereoisomer or solvate thereof in the preparation of a medicament for preventing and / or treating inflammatory diseases.