Trollioside C as well as preparation method and application thereof

Abstract

The invention discloses trollioside C as well as a preparation method and application thereof, and relates to a novel substance with in-vitro anti-inflammatory function as well as a preparation method and application thereof. The invention provides a novel substance with in-vitro anti-inflammatory activity and a preparation method and application thereof. The chemical name of the trolliol glycoside C is maltol-3-1 '-ethoxy hexane-1', 2 ', 3', 4 ', 5', 6 '-hexaol-(4'-2 ')-glucopyranoside, and the molecular formula of the trolliol glycoside C is C21H34O13. The preparation method comprises the following steps: 1, extracting trollius chinensis bunge with ethanol with the volume concentration of 70%; 2, concentrating the extracting solution until no alcohol smell exists, and loading the concentrated extracting solution on a D101 macroporous resin column; and 3, carrying out silica gel column chromatography and gel column chromatography separation on the D101 macroporous resin 30% ethanol elution fraction, and then utilizing high performance liquid chromatography to obtain the trollioside C. The trolliol glycoside C is used for preparing anti-inflammatory drugs. The inhibition rates IC50 of the trollioside C on proinflammatory factors TNF-alpha and IL-6 released by LPS (lipopolysaccharide) stimulated mouse macrophages (RAW264.7) are respectively 22.8 mu mol / L and 31.0 mu mol / L, so that the trollioside C has better in-vitro anti-inflammatory activity. The invention belongs to the field of research on effective components 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, 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 tropaeoside C is C 21 H 34 O 13 , the molecular structure is:

[0008]

[0009] The preparation method of tropaeoside C 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, 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 30% ethanol water elution fraction of 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 dichloromethane and methanol in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the eluate with a volume ratio of dichloromethane to methanol of 100:50 was subjected to gel column chromatography, eluted with methanol, and the eluate was collected to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 35:65 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the elution time of 24 min in the high performance liquid chromatography was collected to obtain tropaeoside C.

[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 high performance liquid chromatography spectrum in step 3 was collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel was used as the filler of the chromatographic column and a Waters 2414 differential detector was used as the detector.

[0015] The tropaeoside C 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 tropaeoside C is used as an active ingredient of an anti-inflammatory drug and is used to inhibit the release of TNF-α and IL-6 in macrophages induced by LPS.

[0019] The chemical name of the tropaeoside C of the present invention is maltol-3-1'-ethoxyhexane-1",2",3",4",5",6"-hexaol-(4'-2")-glucopyranoside, and the molecular formula is C 21H 34 O 13 .

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

[0023] The present invention provides a new compound derived from trollius and a method for extracting, separating and purifying the new compound of the present invention. The new compound is successfully obtained by using ethanol extraction, macroporous adsorption resin, silica gel column chromatography, gel column chromatography and preparative high performance liquid chromatography for separation and purification. The operation method is simple and rapid, and the compound separated by the method has high purity and has anti-inflammatory activity.

[0024] The new compound can inhibit the increase of TNF-α and IL-6 content caused by LPS-induced mouse macrophage RAW264.7 cells. When the compound is at a concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM, the inhibition rate of TNF-α production is 78.5%, 66.3%, 59.7%, 36.6%, and 18.1%, respectively, and the inhibition rate of IL-6 production is 78.5%, 63.8%, 50.8%, 23.2%, and 8.73%, respectively. The IC values ​​of the inhibition rate of the tropaeoside C of the present invention on the proinflammatory factors TNF-α and IL-6 released by mouse macrophages (RAW264.7) stimulated by LPS are as follows: 50 The tropaeoside C of the present invention can be made into an injection, lyophilized powder injection, infusion or oral preparation (including tablets, granules, soft capsules, hard capsules, oral liquids and sustained-release preparations) as an anti-inflammatory drug. 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, tropaeoside C, the molecular formula of tropaeoside C is C 21 H 34 O 13 , the molecular structure is:

[0027]

[0028] Specific embodiment 2: The preparation method of tropaeoside C 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, 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 30% ethanol water elution fraction of 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 dichloromethane and methanol in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the eluate with a volume ratio of dichloromethane to methanol of 100:50 was subjected to gel column chromatography, eluted with methanol, and the eluate was collected to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 35:65 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the elution time of 24 min in the high performance liquid chromatography was collected to obtain tropaeoside C.

[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 high performance liquid chromatography spectrum in step 3 is collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel is used as the filler of the chromatographic column, and Waters 2414 differential detector is used as the detector. Other aspects are the same as those of the second or third specific implementation.

[0034] Specific implementation mode 5: The application of tropaeoside C described in any one of specific implementation modes 1 to 4, wherein the tropaeoside C 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 is different from specific embodiments five to seven in that the tropaeoside C is used as an active ingredient of the anti-inflammatory drug to inhibit the release of TNF-α and IL-6 from macrophages induced by LPS. The rest is the same as specific embodiments five to seven.

[0038] The following experiments were used to verify the effect of the present invention:

[0039] Experiment 1:

[0040] Tropaeoside C, the molecular formula of which is C 21 H 34 O 13 , the molecular structure is:

[0041]

[0042] The preparation method of tropaeoside C 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 30% ethanol water elution fraction of 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 dichloromethane and methanol in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the eluate with a volume ratio of dichloromethane to methanol of 100:50 was subjected to gel column chromatography, eluted with methanol, and the eluate was collected to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 35:65 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the elution time of 24 min in the high performance liquid chromatography was collected to obtain tropaeoside C.

[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 high performance liquid chromatography spectrum in step 3 was collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel was used as the filler of the chromatographic column and a Waters 2414 differential detector was used as the detector.

[0048] The tropaeoside C 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 8.64 mg of nasturtium glycoside C. The new compound was named maltol-3-1'-ethoxyhexane-1",2",3",4",5",6"-hexaol-(4'-2")-glucopyranoside 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 tropaeoside C prepared in Experiment 1 on the production of TNF-α and IL-6 was tested in the following steps:

[0056] 1. Drug preparation

[0057] Tropaeoside C was dissolved in DMSO to prepare a 600 μmol / L stock solution, and diluted with PBS to test concentrations of 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L.

[0058] 2. Cell culture

[0059] 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 the experiment.

[0060] 3. CCK-8 test of the effect of drugs on cell proliferation

[0061] RAW264.7 cells were collected at 2×105 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 C 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. After the culture plate was incubated in an incubator for 45 minutes, the absorbance (OD value) at 450 nm was measured using an enzyme marker. The wells with only CCK-8 solution and complete medium were recorded as complete blank groups, and all groups had three replicates. This was used to determine the safe concentration of tropaeoside C. The results showed that tropaeoside C 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 tropaeoside C 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 the colorimetric 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 the stop solution (2 mol / L H2SO4 solution), mix well, and immediately measure the A450 value. Repeat the above experiment three times in a row. The results showed that when the concentrations were 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L, the inhibition rates were 78.5%, 66.3%, 59.7%, 36.6%, and 18.1%, respectively. IC was calculated using SPSS software. 50 The value was 22.8 μmol / L, indicating that it has good in vitro anti-inflammatory activity.

[0064] 5. Inhibitory activity on IL-6 production

[0065] RAW 264.7 cells were collected at 2×10 5The density of cells / mL was inoculated in a 96-well plate. After attaching to the wall, different concentrations of tropaeoside C and dexamethasone (positive control drug) were used for pre-action for 2 hours, and then 100ng / mL LPS was added to act with the drug for 24 hours. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 10 minutes, and the samples were added to the corresponding wells according to 100μL per well, and 100μL universal diluent was added to the blank wells. After covering the plate sealer, incubate at 37℃ for 60 minutes. Take out the ELISA plate and discard the liquid. Add 100μL of biotinylated antibody working solution to each well, cover the plate sealer and incubate at 37℃ for 60 minutes. Discard the liquid, add 300μL of 1× washing solution to each well, let it stand for 1 minute, shake off the washing solution, and pat dry on absorbent paper. Repeat the washing three times. Add 100μL of enzyme conjugate working solution to each well, cover the plate sealer and incubate at 37℃ for 30 minutes. Discard the liquid, add 300μL 1× washing solution to each well, let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times. Add 90μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37℃ in the dark for 15 min. Take out the ELISA plate, add 50μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450nm. Repeat the above experiment 3 times in a row, and the results show that when the concentrations are 100μmol / L, 50μmol / L, 25μmol / L, 12.5μmol / L, and 6.25μmol / L, the inhibition rates are 78.5%, 63.8%, 50.8%, 23.2%, and 8.73%. IC is calculated using SPSS software. 50 The value was 31.0 μmol / L, indicating that it has good in vitro anti-inflammatory activity.

Claims

1. Tropaeoside C, characterized in that The molecular formula of tropaeoside C is C 21 H 34 O 13 , the molecular structure is:

2. The method for preparing tropaeoside C according to claim 1, characterized in that The preparation method of tropaeoside C 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 turn; 3. The 30% ethanol water elution fraction of 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 dichloromethane and methanol in a volume ratio of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50, and the eluate with a volume ratio of dichloromethane to methanol of 100:50 was subjected to gel column chromatography, eluted with methanol, and the eluate was collected to prepare a high performance liquid chromatography sample, using a mixed solvent of methanol and water in a volume ratio of 35:65 as the mobile phase, the flow rate was 3 mL / min, and the absorption peak appearing at the elution time of 24 min in the high performance liquid chromatography was collected to obtain tropaeoside C.

3. The method for preparing tropaeoside C 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 tropaeoside C according to claim 2, characterized in that The high performance liquid chromatography spectrum in step 3 was collected by using a Waters 2535 semi-preparative high performance liquid chromatograph. 18 Reverse phase silica gel was used as the filler of the chromatographic column and a Waters 2414 differential detector was used as the detector.

5. The use of tropaeoside C according to claim 1, characterized in that The tropaeoside C is used for preparing anti-inflammatory drugs.

6. The use of tropaeoside C 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 tropaeoside 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 use of tropaeoside C according to claim 5, characterized in that The tropaeoside C is used as an active ingredient of an anti-inflammatory drug and is used to inhibit the release of TNF-α and IL-6 in macrophages induced by LPS.

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