Preparation method of tropaeoside B

Abstract

The invention discloses a method for preparing tropaeoside B, and relates to a new substance with in vitro anti-inflammatory activity, a method thereof and an application thereof. The invention provides a new substance with in vitro anti-inflammatory activity, a method thereof and an application thereof. Tropaeoside B has the chemical name 4-[2-(β-D-rhamnosyloxy)ethyl]phenyl-β-D-glucopyranoside, and the molecular formula is C 20 H 30 O 11 . Preparation method: 1. Extract the nasturtium with 70% ethanol by volume; 2. Disperse the recovered ethanol extract with water and apply it to a D101 macroporous resin column; 3. The 30% ethanol elution fraction of the macroporous resin is separated by repeated silica gel column chromatography, gel column chromatography and high performance liquid chromatography to obtain a monomer compound, which is identified as a new compound by spectroscopy and named nasturtium glycoside B. Nautilus glycoside B is used to prepare anti-inflammatory drugs. The IC inhibition rate of nasturtium glycoside B of the present invention on the pro-inflammatory factors TNF-α and IL-Iβ released by mouse macrophages (RAW264.7) stimulated by LPS 50 The results show that the concentrations of 1, 2, and 3 are 35.1 μM and 25.1 μM respectively, indicating that the 1, 2, and 3 have good in vitro anti-inflammatory activity. The invention belongs to the research field of effective ingredients of natural medicines.

Description

Technical Field

[0001] The invention relates to a new substance with in vitro anti-inflammatory effect, a preparation 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, and have inhibitory effects on many bacteria, with small toxic and side effects, making it a very valuable source of plant antibacterial 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 relatively simple. In addition, some studies have shown that its anti-inflammatory activity is not proportional to the total flavonoid content. As a medicinal plant with long-term use as both medicine and food, 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 invention provides a new substance with in vitro anti-inflammatory effect and a method and application thereof.

[0007] Tropaeoside B, wherein the molecular formula of Tropaeoside B is C 20 H 30 O 11 , the molecular structure is:

[0008]

[0009] The preparation method of tropaeoside B is as follows:

[0010] 1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol having a volume concentration of 70% to obtain an 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 30% ethanol water elution fraction 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, 100:5, 100:10, and 100:20, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again. The 100:50 eluate was loaded onto silica gel column chromatography, and eluted with a mixed solvent of ethyl acetate and methanol in a volume ratio of 100:0.625 and 100:1.25, respectively. The eluate in a volume ratio of ethyl acetate to methanol of 100:1.25 was loaded onto hydroxypropyl dextran gel column chromatography, and methanol was used as an eluent. Elution was performed at a rate of 10 drops / min, with 10 mL being an elution fraction. Samples of the 9th to 12th fractions were combined for high performance liquid chromatography preparation, and the absorption peak appearing at the 26th minute of the elution time was collected to obtain tropaeoside B.

[0013] Among them, in step three, the preparative high performance liquid chromatography uses methanol and water in a volume ratio of 35:65 as the mobile phase, and the flow rate is 3 mL / min.

[0014] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction, -0.05 to -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90°C.

[0015] 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.

[0016] The tropaeoside A is used for preparing anti-inflammatory drugs.

[0017] The dosage forms of the anti-inflammatory drug include injection, lyophilized powder injection and oral preparation.

[0018] The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

[0019] The tropaeoside B is used as an active ingredient of an anti-inflammatory drug and is used to inhibit the release of TNF-α and IL-1β from macrophages induced by LPS.

[0020] The chemical name of the tropaeoside B of the present invention is 4-[2-(β-D-rhamnosyloxy)ethyl]phenyl-β-D-glucopyranoside, and the molecular formula is C 20 H 30 O 11 .

[0021] The compounds of the present invention can be added with pharmaceutically acceptable excipients to prepare pharmaceutically acceptable solid preparations or liquid preparations.

[0022] The auxiliary materials described in the present invention are not limited as long as they are pharmaceutically acceptable.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] 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 alcohol extraction, macroporous adsorption resin, silica gel column chromatography, and preparative liquid phase. 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.

[0025] 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 81.60%, 53.54%, 38.75%, 35.53%, and 18.48%, respectively, and the inhibition rate of IL-1β production is 75.49%, 68.77%, 47.85%, 38.25%, and 22.11%, respectively. The IC values ​​of the inhibition rate of nasturtium glycoside B on TNF-α and IL-1β of LPS-induced inflammatory mouse macrophages (RAW264.7) are as follows: 50 The tropaeoside B of the present invention can be prepared 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

[0026] 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. Specific implementation method one:

[0028] In this embodiment, tropaeoside B has a molecular formula of C 20 H 30 O 11 , the molecular structure is:

[0029]

[0030] Specific embodiment 2: The preparation method of tropaeoside B described in specific embodiment 1 is as follows:

[0031] 1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol having a volume concentration of 70% to obtain an extract;

[0032] 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;

[0033] 3. The 30% ethanol water elution fraction 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, 100:5, 100:10, and 100:20, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again. The 100:50 eluate was loaded onto silica gel column chromatography, and eluted with a mixed solvent of ethyl acetate and methanol in a volume ratio of 100:0.625 and 100:1.25, respectively. The eluate in a volume ratio of ethyl acetate to methanol of 100:1.25 was loaded onto hydroxypropyl dextran gel column chromatography, and methanol was used as an eluent. Elution was performed at a rate of 10 drops / min, with 10 mL being an elution fraction. Samples of the 9th to 12th fractions were combined for high performance liquid chromatography preparation, and the absorption peak appearing at the 26th minute of the elution time was collected to obtain tropaeoside B.

[0034] Among them, in step three, the preparative high performance liquid chromatography uses methanol and water in a volume ratio of 35:65 as the mobile phase, and the flow rate is 3 mL / min.

[0035] 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, -0.05 to -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.

[0036] 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.

[0037] Specific embodiment 5: The tropaeoside B described in this embodiment and specific embodiments 1 to 4 is used for preparing anti-inflammatory drugs.

[0038] 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.

[0039] 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.

[0040] Specific embodiment eight: This embodiment is different from specific embodiments one to seven in that the tropaeoside B is used as an active ingredient of the 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.

[0041] The following experiments were used to verify the effect of the present invention:

[0042] Experiment 1:

[0043] Tropaeoside B, wherein the molecular formula of Tropaeoside B is C 20 H 30 O 11 , the molecular structure is:

[0044]

[0045] The preparation method of tropaeoside B is as follows:

[0046] 1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol having a volume concentration of 70% to obtain an extract;

[0047] 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;

[0048] 3. The 30% ethanol water elution fraction 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, 100:5, 100:10, and 100:20, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again. The 100:50 eluate was loaded onto silica gel column chromatography, and eluted with a mixed solvent of ethyl acetate and methanol in a volume ratio of 100:0.625 and 100:1.25, respectively. The eluate in a volume ratio of ethyl acetate to methanol of 100:1.25 was loaded onto hydroxypropyl dextran gel column chromatography, and methanol was used as an eluent. Elution was performed at a rate of 10 drops / min, with 10 mL being an elution fraction. Samples of the 9th to 12th fractions were combined for high performance liquid chromatography preparation, and the absorption peak appearing at the 26th minute of the elution time was collected to obtain tropaeoside B.

[0049] Among them, in step three, the preparative high performance liquid chromatography uses methanol and water in a volume ratio of 35:65 as the mobile phase, and the flow rate is 3 mL / min.

[0050] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction, -0.05 to -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90°C.

[0051] 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.

[0052] This experiment used 6.5 kg of dried nasturtium, and finally obtained 4.47 mg of nasturtium glycoside B. The new compound was named 4-[2-(β-D-rhamnosyloxy)ethyl]phenyl-β-D-glucopyranoside according to its structure. Table 1 shows the NMR data of the new compound: 1 H-NMR and 13 C-NMR in DMSO.

[0053] Table 1: NMR data of the new compounds of the present invention

[0054]

[0055]

[0056] Experiment 2:

[0057] The inhibitory activity of tropaeoside B prepared in Experiment 1 on the production of TNF-α and IL-1β was tested in the following steps:

[0058] 1. Drug preparation: Dissolve tropaeoside B in DMSO to prepare a 600 μmol / L stock solution. Dilute with PBS to test concentrations of 60 μmol / L, 30 μmol / L, 15 μmol / L, 7.5 μmol / L, and 3.75 μmol / L.

[0059] 2. Cell culture

[0060] Mouse macrophages RAW264.7 were grown in DMEM medium containing 10% fetal bovine serum and cultured in suspension in an incubator at 5% CO2, saturated humidity and 37°C. Cells in the logarithmic growth phase were used for the experiment.

[0061] 3. CCK-8 test of the effect of drugs on cell proliferation

[0062] RAW264.7 cells were collected at 2×10 5 The cells were inoculated in a 96-well plate at a density of 100 μg / mL. After the cells adhered to the plate, they were treated with different concentrations of tropaeoside B and dexamethasone (positive control drug) for 24 hours. The supernatant was then removed and discarded. 10 μL CCK-8 solution and 90 μL complete 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 medium were recorded as complete blank groups, and all groups had at least three replicates. This was used to determine the safe concentration of tropaeoside B. The results showed that tropaeoside B had no killing ability on cells at the tested concentration.

[0063] 4. Inhibitory activity on TNF-α production

[0064] RAW 264.7 cells were collected at 2×10 5The 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 reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes. Add 50 μL / well of stop solution (2 mol / L H2SO4 solution), mix well and measure the A450 value immediately. Repeat the above experiment 3 times in a row. The results show that when the concentration is 60 μmol / L, 30 μmol / L, 15 μmol / L, 7.5 μmol / L, and 3.75 μmol / L, the inhibition rate is 81.60%, 53.54%, 38.75%, 35.53%, and 18.48%. IC is calculated using SPSS software 50 The value was 35.1 μM, indicating that it has good in vitro anti-inflammatory activity.

[0065] 5. Inhibitory activity on IL-1β production

[0066] RAW 264.7 cells were collected at 2×10 5The 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 reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes. Add 50 μl / well of stop solution (2 mol / L H2SO4 solution), mix well and measure the A450 value immediately. Repeat the above experiment 3 times in a row. The results show that when the concentration is 60 μmol / L, 30 μmol / L, 15 μmol / L, 7.5 μmol / L, and 3.75 μmol / L, the inhibition rate is 75.49%, 68.77%, 47.85%, 38.25%, and 22.11%. IC is calculated using SPSS software 50 The value was 25.1 μM, indicating that it has good anti-inflammatory activity in vitro.

Claims

1. A method for preparing tropaeoside B, characterized in that The preparation method of tropaeoside B is as follows:

1. placing the nasturtium in an extraction tank of a Soxhlet dynamic extraction and concentration unit, extracting with ethanol having a volume concentration of 70% to obtain an 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 turn; 3. The 30% ethanol water elution fraction 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, 100:5, 100:10, and 100:20, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again, and eluted with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:10, 100:20, 100:30, 100:40, and 100:50, and then the eluate with a volume ratio of dichloromethane to methanol of 100:20 was subjected to silica gel column chromatography again. The eluate with a volume ratio of 100:50 was loaded onto a silica gel column chromatography, and eluted with a mixed solvent of ethyl acetate and methanol in a volume ratio of 100:0.625 and 100:1.25, respectively. The eluate with a volume ratio of ethyl acetate to methanol of 100:1.25 was then loaded onto a hydroxypropyl dextran gel column chromatography, and methanol was used as an eluent. The elution was performed at a rate of 10 drops / min, and each 10 mL was an elution fraction. The samples of the 9th to 12th fractions were combined for high performance liquid chromatography preparation, and the absorption peak appearing at the 26th minute of the elution time was collected to obtain tropaeoside B; In step 3, the preparative high performance liquid chromatography used methanol and water in a volume ratio of 35:65 as the mobile phase, and the flow rate was 3 mL / min; 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; The molecular formula of tropaeoside B is C 20 H 30 O 11 , the molecular structure is:

2. The method for preparing tropaeoside B according to claim 1, characterized in that The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction, -0.05 to -0.08 MPa negative pressure concentration, and the extraction temperature is set to 90°C.

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