Golden lotus glycoside C, its preparation method and application

Abstract

This invention relates to a novel substance with in vitro anti-inflammatory activity, its preparation method, and its application. The chemical name of maltol-3-1'-ethoxyhexane-1”,2”,3”,4”,5”,6”-hexaol-(4'-2”)-glucopyranoside is C. 21 H 34 O 13 Preparation method: 1. Extract *Trollius chinensis* with 70% ethanol (v / v); 2. Concentrate the extract until no alcohol odor remains and load it onto a D101 macroporous resin column; 3. Elute the fraction from the D101 macroporous resin with 30% ethanol, separate by silica gel column chromatography and gel column chromatography, and then obtain *Trollius chinensis* glycoside C using high-performance liquid chromatography. *Trollius chinensis* glycoside C is used to prepare anti-inflammatory drugs. The IC50 of *Trollius chinensis* glycoside C in this invention inhibits the release of pro-inflammatory factors TNF-α and IL-6 from LPS-stimulated mouse macrophages (RAW264.7). 50 The concentrations were 22.8 μmol / L and 31.0 μmol / L, respectively, indicating that it has good in vitro anti-inflammatory activity. This invention belongs to the field of research on effective components of natural drugs.

Description

Technical Field

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

[0002] Golden lotus (Trollius chinensis Bunge) is the dried flower of the golden lotus plant (Trollius L.), belonging to the genus Trollius in the family Ranunculaceae. Its medicinal use was first recorded in the *Compendium of Materia Medica Supplement*. It is cold in nature and bitter in taste, and is believed to have the effects of improving eyesight, clearing heat, and detoxifying. In modern clinical practice, it is mainly used to treat upper respiratory tract infections, tonsillitis, pharyngitis, and otitis media.

[0003] Modern pharmaceutical research shows that the main active ingredients of golden lotus are flavonoids, which have anti-inflammatory, antiviral, and antioxidant effects, with few toxic side effects, making them a valuable source of plant-based anti-inflammatory drugs.

[0004] Currently, there are many preparations of golden lotus on the market, but it has not been included in any edition of the Chinese Pharmacopoeia since the 1977 edition, and the records in some provincial and municipal local standards are also relatively simple. As a medicinal plant with a long history of being used for both food and medicine, systematic chemical separation of golden lotus is of great significance for improving its quality standards and discovering more indicative components with anti-inflammatory activity.

[0005] Inflammation, commonly known as "getting inflamed," is a fundamental pathological process primarily driven by a defensive response in biological tissues in response to stimuli such as trauma or infection. Local manifestations of inflammation include redness, swelling, heat, pain, and impaired function, often accompanied by systemic reactions such as fever and changes in peripheral blood leukocyte counts. Normally, inflammation is beneficial, serving as the body's automatic defense mechanism. However, it can also be harmful, such as attacking the body's own tissues or inflammation occurring in transparent tissues. Therefore, substances that can inhibit inflammatory responses are crucial for protecting public health and improving quality of life. Summary of the Invention

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

[0007] The molecular formula of nasturtium glycoside C is C 21 H 34 O 13 The molecular structural formula is:

[0008]

[0009] The preparation method of the golden lotus glycoside C is carried out according to the following steps:

[0010] 1. Place the golden lotus in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with 70% ethanol by volume to obtain an ethanol extract;

[0011] 2. Concentrate the extract obtained in step 1 until there is no alcohol odor and load it onto a D101 macroporous resin column, then elute with water and 30% ethanol in volume-concentration water sequentially.

[0012] 3. The fraction eluted from step 2 with 30% ethanol and water was separated by silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:3, 100:4, and 100:5. The eluent at a volume ratio of 100:5 was subjected to secondary silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50. The eluent at a volume ratio of 100:50 was subjected to gel column chromatography, eluted with methanol. The eluent was collected to prepare a high-performance liquid chromatography (HPLC) sample. A mixed solvent of methanol and water at a volume ratio of 35:65 was used as the mobile phase at a flow rate of 3 mL / min. The absorption peak appearing at the 24-minute elution time was collected, yielding nasturtium glycoside C.

[0013] The pressure of the Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and -0.08MPa negative pressure concentration, and the extraction temperature is set to 90℃.

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

[0015] The rosiflora glycoside C is used to prepare anti-inflammatory drugs.

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

[0017] The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

[0018] The ginsenoside C, as the active ingredient of the anti-inflammatory drug, is used to inhibit the release of TNF-α and IL-6 from macrophages induced by LPS.

[0019] The chemical name of chrysanthemum glycoside C in this invention is maltol-3-1'-ethoxyhexane-1”,2”,3”,4”,5”,6”-hexaol-(4'-2”)-glucopyranoside, and its molecular formula is C 21H 34 O 13 .

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

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

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

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

[0024] This novel compound inhibits the increase in TNF-α and IL-6 levels induced by LPS in mouse RAW264.7 macrophages. At concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM, the compound exhibited inhibition rates of 78.5%, 66.3%, 59.7%, 36.6%, and 18.1% on TNF-α, and 78.5%, 63.8%, 50.8%, 23.2%, and 8.73% on IL-6, respectively. The IC50 of the inhibitory effect of this invention's strophanthidin C on the release of pro-inflammatory factors TNF-α and IL-6 from LPS-stimulated mouse macrophages (RAW264.7) is also described. 50 The concentrations were 22.8 μM and 31.0 μM, respectively, indicating that it has good in vitro anti-inflammatory activity. The ginsenoside C of this invention can be formulated into injections, lyophilized powder injections, infusions, or oral preparations (including tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations) as an anti-inflammatory drug. Detailed Implementation

[0025] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0026] Specific Implementation Method 1: This implementation method uses chrysanthemum glycoside C, wherein the molecular formula of chrysanthemum glycoside C is C 21 H 34 O 13 The molecular structural formula is:

[0027]

[0028] Specific Implementation Method Two: The preparation method of nasturtium glycoside C described in Specific Implementation Method One is carried out according to the following steps:

[0029] 1. Place the golden lotus in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with 70% ethanol by volume to obtain an ethanol extract;

[0030] 2. Concentrate the extract obtained in step 1 until there is no alcohol odor and load it onto a D101 macroporous resin column, then elute with water and 30% ethanol in volume-concentration water sequentially.

[0031] 3. The fraction eluted from step 2 with 30% ethanol and water was separated by silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:3, 100:4, and 100:5. The eluent at a volume ratio of 100:5 was subjected to secondary silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50. The eluent at a volume ratio of 100:50 was subjected to gel column chromatography, eluted with methanol. The eluent was collected to prepare a high-performance liquid chromatography (HPLC) sample. A mixed solvent of methanol and water at a volume ratio of 35:65 was used as the mobile phase at a flow rate of 3 mL / min. The absorption peak appearing at the 24-minute elution time was collected, yielding nasturtium glycoside C.

[0032] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that the pressure of the Soxhlet dynamic extraction and concentration unit described in step 1 is set to atmospheric pressure extraction and -0.08MPa negative pressure concentration, and the extraction temperature is set to 90℃. Everything else is the same as in Specific Implementation Method 2.

[0033] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods Two or Three in that the high-performance liquid chromatography (HPLC) chromatogram described in step three is acquired using a Waters 2535 semi-preparative HPLC system, with C0.05... 18 The reversed-phase silica gel column is used as the packing material, and a Waters 2414 differential detector is used as the detector. Everything else is the same as in specific embodiments two or three.

[0034] Specific Implementation Method 5: Application of the nasturtium glycoside C described in one of Specific Implementation Methods 1 to 4, wherein the nasturtium glycoside C is used to prepare anti-inflammatory drugs.

[0035] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Five in that the dosage form of the anti-inflammatory drug includes injections, lyophilized powder injections, and oral preparations. Everything else is the same as in Specific Implementation Method Five.

[0036] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Six in that the oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations. Everything else is the same as in Specific Implementation Method Six.

[0037] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods Five to Seven in that the nasturtium glycoside C is used as the active ingredient of an anti-inflammatory drug to inhibit the release of LPS-induced TNF-α and IL-6 from macrophages. Everything else is the same as in Specific Implementation Methods Five to Seven.

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

[0039] Experiment 1:

[0040] Golden lotus glycoside C, wherein the molecular formula of golden lotus glycoside C is C 21 H 34 O 13 The molecular structural formula is:

[0041]

[0042] The preparation method of the golden lotus glycoside C is carried out according to the following steps:

[0043] 1. Place the golden lotus in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract with 70% ethanol by volume to obtain an ethanol extract;

[0044] 2. Concentrate the extract obtained in step 1 until there is no alcohol odor and load it onto a D101 macroporous resin column, then elute with water and 30% ethanol in volume-concentration water sequentially.

[0045] 3. The fraction eluted from step 2 with 30% ethanol and water was separated by silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:3, 100:4, and 100:5. The eluent at a volume ratio of 100:5 was subjected to secondary silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:50. The eluent at a volume ratio of 100:50 was subjected to gel column chromatography, eluted with methanol. The eluent was collected to prepare a high-performance liquid chromatography (HPLC) sample. A mixed solvent of methanol and water at a volume ratio of 35:65 was used as the mobile phase at a flow rate of 3 mL / min. The absorption peak appearing at the 24-minute elution time was collected, yielding nasturtium glycoside C.

[0046] The pressure of the Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and -0.08MPa negative pressure concentration, and the extraction temperature is set to 90℃.

[0047] The high-performance liquid chromatography (HPLC) chromatograms described in step three were acquired using a Waters 2535 semi-preparative HPLC system, with C0.05 as the chromatogram. 18 The reversed-phase silica gel column was used as the packing material, and the Waters 2414 differential detector was used as the detector.

[0048] The rosiflora glycoside C is used to prepare anti-inflammatory drugs.

[0049] The dosage forms of the anti-inflammatory drugs include injections, lyophilized powder injections, and oral preparations.

[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 golden lotus flowers, and finally obtained 8.64 mg of golden lotus glycoside C. The new compound was named maltol-3-1'-ethoxyhexane-1”,2”,3”,4”,5”,6”-hexaol-(4'-2”)-glucopyranoside based on 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 this invention

[0053]

[0054] Experiment 2:

[0055] The inhibitory activity of nasturtium glycoside C prepared in Experiment 1 against TNF-α and IL-6 production was investigated, and the steps were as follows:

[0056] 1. Drug preparation

[0057] Golden lotus glycoside C was dissolved in DMSO to prepare a stock solution of 600 μmol / L. The solution was then 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 experiments.

[0060] 3. Effects of CCK-8 assay on cell proliferation

[0061] RAW264.7 cells were grown at a rate of 2 × 10⁻⁶.5 Cells were seeded at a density of [number] cells / mL in 96-well plates. After adhesion, the plates were treated with different concentrations of thymolica glycoside C and dexamethasone (positive control) for 24 hours. The plates were then removed, the supernatant was discarded, and 10 μL of CCK-8 solution and 90 μL of complete culture medium were added to each well. The plates were incubated for 45 minutes, and the absorbance (OD value) at 450 nm was measured using a microplate reader. Wells containing only CCK-8 solution and complete culture medium were designated as the complete blank group. All groups had three replicates. This was used to determine the safe concentration of thymolica glycoside C. The results showed that thymolica glycoside C had no cell-killing ability at the tested concentration.

[0062] 4. Inhibitory activity against TNF-α production

[0063] RAW 264.7 cells were grown at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates. After adhesion, the cells were pretreated with different concentrations of chrysophanol C and dexamethasone (positive control) for 2 hours, followed by incubation with 100 ng / mL LPS for 24 hours. The required number of pre-coated strips were then removed and placed in 96-well frames. 100 μL of the cell culture supernatant was added to each well. The wells were sealed with sealing film and incubated at room temperature for 120 min. The plates were washed 5 times, with the last wash performed by patting dry on absorbent paper. 100 μL of biotinylated antibody was added to each well. The wells were sealed with clear sealing film and incubated at room temperature for 60 min. The plates were washed 5 times, with the last wash performed by patting dry on absorbent paper. 100 μL of horseradish peroxidase-labeled streptavidin was added to each well. The wells were sealed with white sealing film and incubated at room temperature in the dark for 20 min. The plates were washed 5 times, with the last wash performed by patting dry on absorbent paper. Add 100 μL of TMB chromogenic reagent per well, seal the wells with white sealing film, and incubate at room temperature in the dark for 20 min. Add 50 μL of stop solution (2 mol / L H2SO4 solution) per well, mix well, and immediately measure the A450 value. Repeat the above experiment three times. The results show that the inhibition rates at concentrations of 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L are 78.5%, 66.3%, 59.7%, 36.6%, and 18.1%, respectively. The IC50 value 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 against IL-6 production

[0065] RAW 264.7 cells were grown at a rate of 2 × 10⁻⁶. 5The sample was seeded at a density of 100 μL / mL into a 96-well plate. After adhesion, the plate was pretreated with different concentrations of thymolica glycoside C and dexamethasone (positive control) for 2 hours, followed by incubation with 100 ng / mL LPS for 24 hours. After equilibration at room temperature for 10 minutes, the required strips were removed from the foil bag. 100 μL of sample was added to each well, and 100 μL of universal diluent was added to the blank wells. The plate was sealed and incubated at 37°C for 60 minutes. The plate was then removed and the liquid was discarded. 100 μL of biotinylated antibody working solution was added to each well, and the plate was sealed and incubated at 37°C for 60 minutes. The liquid was discarded, and 300 μL of 1× washing buffer was added to each well. The plate was allowed to stand for 1 minute, the washing buffer was discarded, and the plate was patted dry on absorbent paper. This washing process was repeated 3 times. 100 μL of enzyme conjugate working solution was added to each well, and the plate was sealed and incubated at 37°C for 30 minutes. Discard the liquid, add 300 μL of 1× washing buffer to each well, let stand for 1 min, shake off the washing buffer, and pat dry on absorbent paper. Repeat this washing process 5 times. Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min. Remove the microplate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at 450 nm. Repeat the above experiment 3 times consecutively. The results show that the inhibition rates at concentrations of 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L are 78.5%, 63.8%, 50.8%, 23.2%, and 8.73%, respectively. The IC50 was calculated using SPSS software. 50 The value was 31.0 μmol / L, indicating that it has good in vitro anti-inflammatory activity.

Claims

1. Golden lotus glycoside C, characterized in that... The molecular formula of the rosiflora glycoside C is C 21 H 34 O 13 The molecular structural formula is:

2. The method for preparing tropane glycoside C according to claim 1, characterized in that... The preparation method of the golden lotus glycoside C is carried out according to the following steps:

1. Place the golden lotus in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and extract it with 70% ethanol by volume to obtain an ethanol extract.

2. Concentrate the extract obtained in step 1 until there is no alcohol odor and load it onto a D101 macroporous resin column, then elute with water and 30% ethanol in volume-concentration water sequentially.

3. The fraction eluted from step 2 with 30% ethanol and water was separated by silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:3, 100:4, and 100:

5. The eluent at a volume ratio of 100:5 was subjected to secondary silica gel column chromatography. Eluent was then sequentially eluted with mixed solvents of dichloromethane and methanol at volume ratios of 100:1, 100:2, 100:3, 100:6, 100:12, and 100:

50. The eluent at a volume ratio of 100:50 was subjected to gel column chromatography, eluted with methanol. The eluent was collected to prepare a high-performance liquid chromatography (HPLC) sample. A mixed solvent of methanol and water at a volume ratio of 35:65 was used as the mobile phase at a flow rate of 3 mL / min. The absorption peak appearing at the 24-minute elution time was collected, yielding nasturtium glycoside C.

3. The method for preparing tropane glycoside C according to claim 2, characterized in that... The pressure of the Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and -0.08MPa negative pressure concentration, and the extraction temperature is set to 90℃.

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

5. The application of the chrysanthemum glycoside C according to claim 1, characterized in that... The rosiflora glycoside C is used to prepare anti-inflammatory drugs.

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

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

8. The application of chrysanthemum glycoside C according to claim 5, characterized in that... The ginsenoside C, as the active ingredient of the anti-inflammatory drug, is used to inhibit the release of TNF-α and IL-6 from macrophages induced by LPS.

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