Tropaeolum phenol A and its preparation method and application
Through systematic extraction and separation methods, analotus A was successfully obtained. This compound has significant anti-inflammatory activity, solving the shortcomings in the research on the quality standards and anti-inflammatory components of the prior art, and achieving efficient inflammatory inhibition effect.
Patent Information
- Application Number
- CN202310872116.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-17
- Publication Date
- 2025-02-11
- Estimated Expiration
- 2043-07-17
AI Technical Summary
The prior art is difficult to effectively improve the quality standards of the genus Lotus and discover more anti-inflammatory active ingredients, and its collection in the pharmacopoeia is relatively simple and lacks systematic chemical separation research.
The new compound analociol A was successfully isolated and purified by ethanol extraction of Soxhlet dynamic extraction concentration unit, macroporous resin column elution, silica gel column chromatography and high performance liquid chromatography.
Anambalum A has significant in vitro anti-inflammatory activity, can inhibit the release of TNF-α and IL-1β by LPS-induced mouse macrophages, and has a low IC50 value, indicating that it has high efficacy in inhibiting inflammatory responses. .
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Abstract
Description
Technical Field
[0001] The present invention relates to a novel substance with in vitro anti-inflammatory effect and a method and application thereof. Background Art
[0002] Trollius chinensis Bunge is the dried flower of the plant Trollius L. of the Ranunculaceae family. Its medicinal use was first recorded in the "Supplement to Compendium of Materia Medica". It is cold in nature, bitter in taste, and has the effects of improving eyesight, clearing away heat and detoxifying. In modern clinical practice, it is mainly used to treat upper respiratory tract infections, tonsillitis, pharyngitis, otitis media, etc.
[0003] Modern pharmaceutical research shows that the main active ingredients of Nasturtium are flavonoids, which have anti-inflammatory, antiviral, antioxidant and other effects, with small toxic and side effects, and are a very valuable source of plant anti-inflammatory drugs.
[0004] At present, there are many preparations of Nasturtium in the market, but it has not been included in the 1977 edition of the Chinese Pharmacopoeia, and the records in the local standards of some provinces and cities are also relatively simple. As a medicinal plant with long-standing medicinal and edible properties, it is of great significance to systematically chemically separate Nasturtium in order to improve its quality standards and discover more index components with anti-inflammatory activity.
[0005] Inflammation is what we usually call "inflammation". It is a basic pathological process that occurs when biological tissues are stimulated by certain damaging factors such as trauma, infection, etc., which is mainly a defensive response. The local manifestations of inflammation are redness, swelling, heat, pain and dysfunction, and are also accompanied by systemic reactions such as fever and changes in peripheral white blood cell counts. Under normal circumstances, inflammation is beneficial and is an automatic defensive response of the human body. However, sometimes inflammation is also harmful, such as attacks on the body's own tissues, inflammation occurring in transparent tissues, and so on. Therefore, substances that can inhibit inflammatory responses are of great significance to protecting people's health and improving their quality of life. Summary of the invention
[0006] The purpose of the present invention is to provide a new compound with in vitro anti-inflammatory activity and a preparation method and application thereof.
[0007] The molecular formula of tropaeolum phenol A is C 9 H 10 O 4 , the molecular structure is:
[0008]
[0009] The preparation method of tropaeolum phenol A is carried out according to the following steps:
[0010] 1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and extracting with ethanol having a volume concentration of 70% to obtain an ethanol extract;
[0011] 2. The extract obtained in step 1 was concentrated until it was free of alcohol and then loaded onto a D101 macroporous resin column, followed by elution with water and 30% ethanol in turn;
[0012] 3. The fraction eluted with 30% ethanol water by volume in step 2 was subjected to silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:3, 100:4, and 100:5, and the eluate with a volume ratio of dichloromethane to methanol of 100:5 was subjected to secondary silica gel column chromatography, and eluted with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the petroleum ether was taken. The eluate with a volume ratio of 100:50 to ethyl acetate was separated by silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:0.625 and 100:1.25 in sequence, and the eluate with a volume ratio of dichloromethane to methanol of 100:1.25 was collected for high performance liquid chromatography preparation, using a mixed solvent of methanol and water in a volume ratio of 38:62 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the 8th minute of the elution time was collected to obtain tropaeolum phenol A.
[0013] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction and -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90°C.
[0014] The preparative HPLC in step 3 uses a Waters 2535 semi-preparative HPLC, the detector is a Waters 2414 differential detector, and the chromatographic column is filled with C 18 Reversed phase silica gel.
[0015] The tropaeolum phenol A is used for preparing anti-inflammatory drugs.
[0016] The dosage forms of the anti-inflammatory drug include injection, lyophilized powder injection and oral preparation.
[0017] The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.
[0018] The tropaeolum phenol A is used as an active ingredient of an anti-inflammatory drug to inhibit the release of TNF-α and IL-1β from macrophages induced by LPS.
[0019] The chemical name of the nasturtium phenol A of the present invention is 1-(3,4-dihydroxyphenyl)-3-hydroxypropan-2-one, and the molecular formula is C9 H 10 O 4 .
[0020] The compounds of the present invention can be added with pharmaceutically acceptable excipients to prepare pharmaceutically acceptable solid preparations or liquid preparations.
[0021] The auxiliary materials described in the present invention are not limited as long as they are pharmaceutically acceptable.
[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0023] The present invention provides a new compound derived from nasturtium and a method for extracting, separating and purifying the new compound of the present invention. The new compound is successfully obtained by separation and purification using methods such as ethanol extraction, macroporous adsorption resin, silica gel column chromatography, and preparative high performance liquid chromatography. The operation method is simple and rapid, and the compound separated by the method has a high purity. In addition, studies have shown that the above compound has anti-inflammatory activity.
[0024] The new compound can inhibit the increase of TNF-α and IL-1β content caused by LPS-induced mouse macrophage RAW264.7 cells. When the compound is at a concentration of 60μM, 30μM, 15μM, 7.5μM, and 3.75μM, the inhibition rate of TNF-α production is 81.6%, 53.5%, 38.8%, 35.5%, and 18.5%, respectively, and the inhibition rate of IL-1β production is 75.5%, 68.8%, 47.8%, 38.3%, and 22.1%, respectively. The IC inhibition rate of the nasturtium phenol A of the present invention on the proinflammatory factors TNF-α and IL-1β released by mouse macrophages (RAW264.7) stimulated by LPS is 50 The nasturtium phenol A of the present invention can be made into anti-inflammatory drugs such as injection, lyophilized powder injection, infusion or oral preparation (including tablets, granules, soft capsules, hard capsules, oral liquids and sustained-release preparations). DETAILED DESCRIPTION
[0025] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.
[0026] Specific implementation method 1: In this implementation method, the molecular formula of tropaeolum phenol A is C 9 H 10 O 4 , the molecular structure is:
[0027]
[0028] Specific embodiment 2: The preparation method of tropaeolum phenol A described in specific embodiment 1 is carried out according to the following steps:
[0029] 1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and extracting with ethanol having a volume concentration of 70% to obtain an ethanol extract;
[0030] 2. The extract obtained in step 1 was concentrated until it was free of alcohol and then loaded onto a D101 macroporous resin column, followed by elution with water and 30% ethanol in turn;
[0031] 3. The fraction eluted with 30% ethanol water by volume in step 2 was subjected to silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:3, 100:4, and 100:5, and the eluate with a volume ratio of dichloromethane to methanol of 100:5 was subjected to secondary silica gel column chromatography, and eluted with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the petroleum ether was taken. The eluate with a volume ratio of 100:50 to ethyl acetate was separated by silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:0.625 and 100:1.25 in sequence, and the eluate with a volume ratio of dichloromethane to methanol of 100:1.25 was collected for high performance liquid chromatography preparation, using a mixed solvent of methanol and water in a volume ratio of 38:62 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the 8th minute of the elution time was collected to obtain tropaeolum phenol A.
[0032] Specific implementation method three: This implementation method is different from specific implementation method two in that the pressure of the Soxhlet dynamic extraction and concentration unit in step one is set to normal pressure extraction, -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90° C. The rest is the same as specific implementation method two.
[0033] Specific embodiment 4: This embodiment is different from specific embodiment 2 or 3 in that the preparative high performance liquid chromatography described in step 3 uses a Waters 2535 semi-preparative high performance liquid phase, the detector is a Waters 2414 differential detector, and the filler of the chromatographic column is C 18 Reverse phase silica gel. Others are the same as those in the second or third embodiment.
[0034] Specific embodiment 5: The tropaeolum ol A described in this embodiment and specific embodiments 1 to 4 is used for preparing anti-inflammatory drugs.
[0035] Specific embodiment 6: This embodiment is different from the specific embodiment 5 in that the dosage form of the anti-inflammatory drug includes injection, lyophilized powder injection, and oral preparation. The rest is the same as the specific embodiment 5.
[0036] Specific embodiment 7: This embodiment is different from specific embodiment 6 in that the oral preparation includes tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations. The rest is the same as specific embodiment 6.
[0037] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that the tropaeolum phenol A is used as an active ingredient of an anti-inflammatory drug to inhibit the release of TNF-α and IL-1β from macrophages induced by LPS. The rest is the same as specific embodiments one to seven.
[0038] The following experiments were used to verify the effect of the present invention:
[0039] Experiment 1:
[0040] Tropaeolum phenol A, wherein the molecular formula of the Tropaeolum phenol A is C 9 H 10 O 4 , the molecular structure is:
[0041]
[0042] The preparation method of tropaeolum phenol A is carried out according to the following steps:
[0043] 1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and extracting with ethanol having a volume concentration of 70% to obtain an ethanol extract;
[0044] 2. The extract obtained in step 1 was concentrated until it was free of alcohol and then loaded onto a D101 macroporous resin column, followed by elution with water and 30% ethanol in turn;
[0045] 3. The fraction eluted with 30% ethanol water by volume in step 2 was subjected to silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:3, 100:4, and 100:5, and the eluate with a volume ratio of dichloromethane to methanol of 100:5 was subjected to secondary silica gel column chromatography, and eluted with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the petroleum ether was taken. The eluate with a volume ratio of 100:50 to ethyl acetate was separated by silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:0.625 and 100:1.25 in sequence, and the eluate with a volume ratio of dichloromethane to methanol of 100:1.25 was collected for high performance liquid chromatography preparation, using a mixed solvent of methanol and water in a volume ratio of 38:62 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the 8th minute of the elution time was collected to obtain tropaeolum phenol A.
[0046] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction and -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90°C.
[0047] The preparative HPLC in step 3 uses a Waters 2535 semi-preparative HPLC, the detector is a Waters 2414 differential detector, and the chromatographic column is filled with C 18 Reversed phase silica gel.
[0048] The tropaeolum phenol A is used for preparing anti-inflammatory drugs.
[0049] The dosage forms of the anti-inflammatory drug include injection, lyophilized powder injection and oral preparation.
[0050] The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.
[0051] This experiment used 6.5 kg of dried nasturtium, and finally obtained 4.07 mg of nasturtium phenol A. The new compound was named 1-(3,4-dihydroxyphenyl)-3-hydroxypropan-2-one according to its structure. Table 1 shows the NMR data of the new compound: 1 H-NMR and 13 C-NMR in DMSO.
[0052] Table 1: NMR data of the new compounds of the present invention
[0053]
[0054] Experiment 2:
[0055] The inhibitory activity of tropaeolum phenol A prepared in Experiment 1 on the production of TNF-α and IL-1β was tested in the following steps:
[0056] 1. Drug preparation
[0057] Tropaeolum phenol A was dissolved in DMSO to prepare a 600 μmol / L stock solution, and diluted with PBS to test concentrations of 60 μmol / L, 30 μmol / L, 15 μmol / L, 7.5 μmol / L, and 3.75 μmol / L.
[0058] 2. Cell culture
[0059] Mouse macrophage RAW264.7 cells were grown in DMEM containing 10% fetal bovine serum and in an atmosphere of 5% CO. 2 The cells were suspended and cultured in an incubator at 37°C and saturated humidity, and the cells in the logarithmic growth phase were used for the experiment.
[0060] 3. CCK-8 test of the effect of drugs on cell proliferation
[0061] RAW264.7 cells were collected at 2×10 5 The cells were inoculated at a density of 100 μg / mL in a 96-well plate. After adhering to the wall, they were treated with different concentrations of nasturtium phenol A and dexamethasone (positive control drug) for 24 hours. The supernatant was then removed and discarded. 10 μL of CCK-8 solution and 90 μL of complete culture medium were added to each well. The culture plate was incubated in an incubator for 45 minutes, and the absorbance (OD value) at 450 nm was measured with an enzyme marker. The wells with only CCK-8 solution and complete culture medium were recorded as complete blank groups, and all groups had at least three replicate wells. The safe action concentration of nasturtium phenol A was determined in this way. The results showed that nasturtium phenol A had no killing ability on cells at the tested concentration.
[0062] 4. Inhibitory activity on TNF-α production
[0063] RAW 264.7 cells were collected at 2×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. After the cells adhered to the wall, they were pre-treated with different concentrations of nasturtium phenol A and dexamethasone (positive control drug) for 2 hours, and then 100 ng / mL LPS was added to act with the drug for 24 hours. The number of pre-coated strips required for the experiment was taken out and placed in a 96-well frame. 100 μL of the above cell culture supernatant was added to the corresponding wells, and the reaction wells were sealed with a sealing film. Incubated at room temperature for 120 minutes. Wash the plate 5 times, and place it on thick absorbent paper and pat dry for the last time. Add 100 μL / well of biotinylated antibody. Seal the reaction wells with a sealing film (transparent) and incubate at room temperature for 60 minutes. Wash the plate 5 times, and place it on thick absorbent paper and pat dry for the last time. Add 100 μL / well of horseradish peroxidase-labeled Streptavidin. Seal the reaction wells with a sealing film (white) and incubate at room temperature in the dark for 20 minutes. Wash the plate 5 times, and place it on thick absorbent paper and pat dry for the last time. Add 100 μL / well of TMB solution, seal the wells with a plate sealer (white), and incubate at room temperature in the dark for 20 minutes. Add stop solution (2 mol / L H 2 SO 4 Solution) 50μL / well, mix well and measure A450 value immediately. Repeat the above experiment 3 times. The results show that the inhibition rate is 81.6%, 53.5%, 38.8%, 35.5% and 18.5% when the concentration is 60μmol / L, 30μmol / L, 15μmol / L, 7.5μmol / L and 3.75μmol / L. SPSS software is used to calculate IC 50 The value was 18.8 μM, indicating that it has good in vitro anti-inflammatory activity.
[0064] 5. Inhibitory activity on IL-1β production
[0065] RAW 264.7 cells were collected at 2×105 The density of cells / mL was inoculated in a 96-well plate. After the cells adhered to the wall, different concentrations of nasturtium phenol A and dexamethasone (positive control drug) were used for pre-treatment for 2 hours, and then 100ng / mL LPS was added to act with the drug for 24 hours. The number of pre-coated strips required for the experiment was taken out and placed in the 96-well frame. The samples or different concentrations of standard substances were added to the corresponding wells at 100μl / well. The reaction wells were sealed with a sealing film (transparent) and incubated at room temperature for 120 minutes. The plate was washed 5 times, and the last time it was placed on thick absorbent paper and patted dry. Biotinylated antibody was added at 100μl / well. The reaction wells were sealed with a sealing film (transparent) and incubated at room temperature for 60min. The plate was washed 5 times, and the last time it was placed on thick absorbent paper and patted dry. Horseradish peroxidase-labeled Streptavidin was added at 100μl / well. The reaction wells were sealed with a sealing film (white) and incubated at room temperature in the dark for 20min. The plate was washed 5 times, and the last time it was placed on thick absorbent paper and patted dry. Add 100 μl / well of TMB solution, seal the wells with a plate sealer (white), and incubate at room temperature in the dark for 20 min. Add stop solution (2 mol / L H 2 SO 4 Solution) 50μl / well, mix well and measure A450 value immediately. Repeat the above experiment 3 times. The results show that the inhibition rate is 75.5%, 68.8%, 47.8%, 38.3% and 22.1% when the concentration is 60μmol / L, 30μmol / L, 15μmol / L, 7.5μmol / L and 3.75μmol / L. SPSS software is used to calculate IC 50 The value was 14.6 μM, indicating that it has good in vitro anti-inflammatory activity.
Claims
1. Tropaeolum phenol A, characterized in that The molecular formula of tropaeolum phenol A is C9H 10 O4, molecular structure formula is: 。 2. The method for preparing tropaeolum phenol A according to claim 1, characterized in that The preparation method of tropaeolum phenol A is carried out according to the following steps:
1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and extracting with ethanol having a volume concentration of 70% to obtain an ethanol extract; 2. The extract obtained in step 1 was concentrated until it was free of alcohol and then loaded onto a D101 macroporous resin column, followed by elution with water and 30% ethanol in volume concentration in sequence; 3. The fraction eluted with 30% ethanol water by volume in step 2 was subjected to silica gel column chromatography, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:3, 100:4, and 100:5, and the eluate with a volume ratio of dichloromethane to methanol of 100:5 was subjected to secondary silica gel column chromatography, and a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50 was used. The mixture was eluted in sequence, and the eluate with a volume ratio of petroleum ether to ethyl acetate of 100:50 was subjected to silica gel column chromatography separation. The mixture was eluted in sequence with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:0.625 and 100:1.
25. The eluate with a volume ratio of dichloromethane to methanol of 100:1.25 was collected for high performance liquid chromatography preparation, and a mixed solvent of methanol and water in a volume ratio of 38:62 was used as the mobile phase at a flow rate of 3 mL / min. The absorption peak appearing at the 8th min of the elution time was collected to obtain tropaeolum phenol A.
3. The method for preparing tropaeolum phenol A according to claim 2, characterized in that The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction and -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90°C.
4. The method for preparing tropaeolum phenol A according to claim 2, characterized in that The high performance liquid chromatography in step 3 adopts Waters 2535 semi-preparative high performance liquid chromatography, the detector is Waters 2414 differential detector, and the filler of the chromatographic column is C 18 Reversed phase silica gel.
5. The use of tropaeolum phenol A according to claim 1, characterized in that The tropaeolum phenol A is used for preparing anti-inflammatory drugs.
6. The use of tropaeolum phenol A according to claim 5, characterized in that The dosage forms of the anti-inflammatory drug include injection, lyophilized powder injection and oral preparation.
7. The use of tropaeolum phenol A 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 use of tropaeolum phenol A according to claim 5, characterized in that The tropaeolum phenol A is used as an active ingredient of an anti-inflammatory drug to inhibit the release of TNF-α and IL-1β from macrophages induced by LPS.