Preparation method and application of daylily lactone A
By isolating and purifying hemerocallis lactone A from daylily flowers, the problem of the lack of effective neuroprotective drugs in the existing technology has been solved, the survival rate of H2O2-induced HT22 cells has been improved, and a variety of neuroprotective drug formulations have been provided.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2026-04-03
AI Technical Summary
There is a lack of effective neuroprotective drugs in the current technology, especially for nerve cell damage caused by oxidative stress, and there is a lack of compounds with neuroprotective activity extracted from daylily flowers.
Hemerocallis lactone A was isolated and purified from daylily flowers using ethanol extraction, macroporous adsorption resin, silica gel column chromatography, hydroxypropyl dextran gel column chromatography, and preparative liquid chromatography, and was then formulated into a neuroprotective drug.
Hemerocallis lactone A significantly improves the survival rate of H2O2-induced HT22 cells in vitro, exhibiting good neuroprotective activity, and is suitable for the preparation of various dosage forms such as injections, lyophilized powder injections, and oral preparations.
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Abstract
Description
Technical Field
[0001] This invention relates to a novel substance with in vitro neuroprotective activity, its method and application. Background Technology
[0002] Daylily flowers (also known as golden needles or yellow flowers) are the dried flower buds of the lemon daylily (Hemerocallis citrina Baroni), a plant belonging to the genus Hemerocallis in the family Liliaceae. Recorded in the *Compendium of Materia Medica*, daylily flowers are described as having a cool nature, sweet taste, and no toxicity. They are believed to have effects such as clearing damp-heat, relieving chest congestion, calming the five internal organs, promoting happiness, and improving lightness of body and eyesight. They can be used to treat symptoms such as painful urination, feverishness, and jaundice. Modern pharmacological studies have shown that they possess antidepressant, anti-inflammatory, antioxidant, and neuroprotective effects.
[0003] Modern pharmaceutical research shows that the main active ingredients of daylily flowers are flavonoids, phenolic acids, fatty acids, anthraquinones, dihydrozirconiums, etc., which have antidepressant, neuroprotective, anti-inflammatory, and antioxidant effects, with few toxic side effects, making them a valuable source of phytoneuroprotective drugs.
[0004] Daylily flowers are recognized as a food product under geographical indication standards in Qidong, Dali, and Huaiyang. As a long-established medicinal and edible plant, systematic chemical separation of daylily flowers is of great significance for improving its quality standards and discovering more indicative components with neuroprotective activity.
[0005] Oxidative stress refers to a pathological state in which the balance between the body's oxidative and antioxidant systems is disrupted when the body is subjected to various harmful stimuli, resulting in an excessive production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), exceeding the cellular antioxidant defense mechanism's ability to eliminate them, thus leading to cell and tissue damage. Neuroprotection refers to strategies that use various means (such as drug intervention, lifestyle modifications, and physical therapy) to prevent, delay, or reduce nerve cell damage and halt the decline of neurological function. Its aim is to maintain the normal structure and function of the nervous system, especially targeting vulnerable neuronal populations, such as hippocampal neurons (closely related to learning and memory) and substantia nigra neurons (prone to damage in Parkinson's disease, affecting motor function regulation). HT22 cells are a cell line derived from mouse hippocampal neurons; the hippocampus plays a crucial role in neural functions such as learning and memory. By using the H2O2-induced HT22 cell damage model, compounds that can effectively protect cells from damage can be screened out. Further in-depth pharmacological and toxicological studies on these compounds may lead to the development of novel neuroprotective drugs, providing more treatment options for the clinical treatment of neurological diseases. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing a new compound with in vitro neuroprotective activity and its application.
[0007] Hemerocallis lactone A, wherein the molecular formula of hemerocallis lactone A is C 17 H 28 O5, the molecular structural formula is:
[0008]
[0009] The preparation method of the daylily lactone A is as follows:
[0010] 1. Place the daylily flowers in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold extraction with 80% ethanol (volume concentration) for 72 hours each time, for 3 extractions, and combine the extracts to obtain the extract;
[0011] 2. Concentrate the extract obtained in step 1 until there is no alcohol odor, dilute with water, and then load it onto a D101 macroporous resin column. Then, elute with water and water with volume concentrations of 15%, 30%, 50%, 70%, and 90% ethanol in sequence.
[0012] 3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using mixed solvents of dichloromethane and methanol at volume ratios of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, respectively. Take the eluent with a dichloromethane to methanol volume ratio of 100:20 and load it onto a hydroxypropyl dextran gel column for chromatography, using methanol as the eluent at a rate of 1.5 mL / min. Each elution fraction is 15 mL. When the color band descends to above the column outlet, start collecting the fractions. Combine the samples of fractions 16-18 to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak that appears at a retention time of 22.5 min in the HPLC, which is the daylily lactone A.
[0013] The Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and ambient temperature extraction.
[0014] Step 3 describes the preparation using a Waters 2535 semi-preparative HPLC system, with a Waters 2998 DAD detector as the detector and C0 as the reference. 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water at a volume ratio of 42:58, with a flow rate of 3 ml / min.
[0015] The daylily lactone A is used to prepare neuroprotective drugs.
[0016] The dosage forms of the neuroprotective 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 daylily lactone A, as an active ingredient in neuroprotective drugs, is used to improve the survival rate of H2O2-induced HT22 cell damage.
[0019] The chemical name of the daylily lactone A of this invention is 11-(2-hydroxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl) undecanoic acid, and its molecular formula is C2. 17 H 28 O5.
[0020] The present invention provides that daylily lactone A can be formulated into pharmaceutically acceptable solid or liquid preparations 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 daylily flowers and a method for extracting, separating, and purifying the novel compound. The method employs ethanol extraction, macroporous adsorption resin, silica gel column chromatography, hydroxypropyl dextran gel column chromatography, and preparative 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. Furthermore, studies have shown that the above compound has neuroprotective activity.
[0024] This novel compound can improve the survival rate of H2O2-induced HT22 cell damage. At concentrations of 50 μM, 25 μM, 12.5 μM, and 0 μM, the survival rates of H2O2-induced HT22 cell damage were 81.46%, 75.60%, 71.16%, and 63.77%, respectively, indicating good in vitro neuroprotective activity. The hemerocallis lactone A of this invention can be formulated into injections, lyophilized powder for injection, infusions, or oral preparations (including tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release formulations) as neuroprotective drugs. 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 daylily lactone A, wherein the molecular formula of daylily lactone A is C 17 H 28 O5, the molecular structural formula is:
[0027]
[0028] Specific Implementation Method Two: The preparation method of daylily lactone A described in Specific Implementation Method One is as follows:
[0029] 1. Place the daylily flowers in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold extraction with 80% ethanol (volume concentration) for 72 hours each time, for 3 extractions, and combine the extracts to obtain the extract;
[0030] 2. Concentrate the extract obtained in step 1 until there is no alcohol odor, dilute with water, and then load it onto a D101 macroporous resin column. Then, elute with water and water with volume concentrations of 15%, 30%, 50%, 70%, and 90% ethanol in sequence.
[0031] 3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using mixed solvents of dichloromethane and methanol at volume ratios of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, respectively. Take the eluent with a dichloromethane to methanol volume ratio of 100:20 and load it onto a hydroxypropyl dextran gel column for chromatography, using methanol as the eluent at a rate of 1.5 mL / min. Each elution fraction is 15 mL. When the color band descends to above the column outlet, start collecting the fractions. Combine the samples of fractions 16-18 to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak that appears at a retention time of 22.5 min in the HPLC, which is the daylily lactone A.
[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 the extraction temperature is room temperature. 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) preparation described in step three uses a Waters 2535 semi-preparative HPLC system, with a Waters 2998 DAD detector as the detector, and C00 as the reference. 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water at a volume ratio of 42:58, with a flow rate of 3 ml / min. Other aspects were the same as in specific embodiments two or three.
[0034] Specific Implementation Method 5: The daylily lactone A described in Specific Implementation Methods 1 to 4 is used to prepare neuroprotective drugs.
[0035] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Five in that the dosage forms of the neuroprotective drugs include 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 One to Seven in that the daylily lactone A is used as the active ingredient of a neuroprotective drug to improve the survival rate of H2O2-induced HT22 cell damage. Everything else is the same as in Specific Implementation Methods One to Seven.
[0038] The following experiments were used to verify the effectiveness of the invention:
[0039] Experiment 1:
[0040] Hemerocallis lactone A, wherein the molecular formula of hemerocallis lactone A is C 17 H 28 O5, the molecular structural formula is:
[0041]
[0042] The preparation method of the daylily lactone A is as follows:
[0043] 1. Place the daylily flowers in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold extraction with 80% ethanol (volume concentration) for 72 hours each time, for 3 extractions, and combine the extracts to obtain the extract;
[0044] 2. Concentrate the extract obtained in step 1 until there is no alcohol odor, dilute with water, and then load it onto a D101 macroporous resin column. Then, elute with water and water with volume concentrations of 15%, 30%, 50%, 70%, and 90% ethanol in sequence.
[0045] 3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using mixed solvents of dichloromethane and methanol at volume ratios of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, respectively. Take the eluent with a dichloromethane to methanol volume ratio of 100:20 and load it onto a hydroxypropyl dextran gel column for chromatography, using methanol as the eluent at a rate of 1.5 mL / min. Each elution fraction is 15 mL. When the color band descends to above the column outlet, start collecting the fractions. Combine the samples of fractions 16-18 to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak that appears at a retention time of 22.5 min in the HPLC, which is the daylily lactone A.
[0046] The Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and ambient temperature extraction.
[0047] Step 3 describes the preparation using a Waters 2535 semi-preparative HPLC system, with a Waters 2998 DAD detector as the detector and C0 as the reference. 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water at a volume ratio of 42:58, with a flow rate of 3 ml / min.
[0048] This experiment used 10.0 kg of dried daylily flowers, ultimately obtaining 5.27 mg of daylily lactone A. The new compound was named 11-(2-hydroxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl)undecanoic acid based on its structure. Table 1 shows the NMR data of the new compound. 1 H-NMR and 13 C-NMR in CD3OD.
[0049] Table 1: NMR data of daylily lactone A of the present invention
[0050]
[0051] Experiment 2:
[0052] The daylily lactone A prepared in Experiment 1 was used to improve the survival rate of H2O2-induced HT22 cell damage. The steps are as follows:
[0053] 1. Drug preparation: Hemerocallis lactone A was dissolved in DMSO to prepare a 50 mmol / L stock solution. It was then diluted with PBS to test concentrations of 50 μmol / L, 25 μmol / L, and 12.5 μmol / L.
[0054] 2. Cell Culture
[0055] Mouse hippocampal neuronal HT22 cells 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.
[0056] 3. CCK-8 assay for the effect of daylily lactone A on cell proliferation
[0057] Mouse hippocampal neuronal HT22 cells were used at 2×10 4Cells were seeded at a density of [number] cells / well in 96-well plates. After adhesion, the cells were treated with different concentrations of daylily lactone A for 24 h. The plates were then removed, the supernatant discarded, and 100 μL of complete culture medium containing 10% CCK-8 cells was added to each well. The 96-well plates were incubated for 30 min, 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 control group. All groups had three replicates. This was used to determine the safe concentration of daylily lactone A. The results showed that daylily lactone A had no cell-killing ability at the tested concentration.
[0058] 4. CCK-8 assay to protect hemerocallis lactone A from H2O2-induced cell damage model
[0059] Healthy HT22 cells were seeded into sterile 96-well plates at a density of 2 × 10⁻⁶ cells / well. 4 Cells were added to each well with 100 μL of complete culture medium. The mixture was incubated overnight in a cell culture incubator. When the cells reached 70-80% of the bottom area of the 96-well plate, the plate was removed, and the supernatant was removed using a pipette. 100 μL of cell culture medium was added to the blank control and model groups. Cell culture medium containing 50 μmol / L, 25 μmol / L, and 12.5 μmol / L of daylily lactone A was added to the drug-treated groups, respectively. Three replicates were set up for each group, and the wells were incubated for another 2 hours. After 24 hours, the cell supernatant was aspirated. Except for the blank control wells, complete culture medium containing 50 μmol / L H2O2 was added to each well, mixed, and incubated for another 2 hours. After the treatment was completed, CCK-8 solution was added to each well to test the survival rate of each group relative to the blank control group. The results showed that when the compound concentration was 50 μmol / L, 25 μmol / L, and 12.5 μmol / L, the survival rates of H2O2-induced HT22 cell damage were 81.46%, 75.60%, and 71.16%, respectively, while the cell survival rate of the model group without the drug was 63.77%, indicating that Hemerocallis lactone A has good in vitro neuroprotective activity.
Claims
1. A method for preparing daylily lactone A, characterized in that... The preparation method of the daylily lactone A is as follows:
1. Place the daylily flowers in the extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold extraction with 80% ethanol (volume concentration) for 72 hours each time, for 3 extractions, and combine the extracts to obtain the extract; 2. Concentrate the extract obtained in step 1 until there is no alcohol odor, dilute with water, and then load it onto a D101 macroporous resin column. Then, elute with water and water with volume concentrations of 15%, 30%, 50%, 70%, and 90% ethanol in sequence.
3. Separate the 50% volume concentration elution fraction from step 2 by silica gel column chromatography. Elute sequentially with a mixed solvent of dichloromethane and methanol at volume ratios of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:
100. Take the eluent with a dichloromethane to methanol volume ratio of 100:20 and precipitate it onto a hydroxypropyl dextran gel column using methanol as the eluent at a rate of 1.5 mL / min. Each elution fraction is 15 mL. Collect the fractions when the color band descends to above the column outlet. Combine the samples of fractions 16-18 to prepare a high performance liquid chromatography (HPLC) sample. Collect the absorption peak that appears at a retention time of 22.5 min in the HPLC, which is the daylily lactone A.
2. The method for preparing daylily lactone A according to claim 1, characterized in that... The Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and ambient temperature extraction.
3. The method for preparing daylily lactone A according to claim 1, characterized in that... Step 3 describes the preparation using a Waters 2535 semi-preparative HPLC system, with a Waters 2998 DAD detector as the detector and C0 as the reference. 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water at a volume ratio of 42:58, with a flow rate of 3 ml / min.
4. The application of daylily lactone A according to claim 1, characterized in that... The daylily lactone A is used to prepare neuroprotective drugs.
5. The application of daylily lactone A according to claim 4, characterized in that... The dosage forms of the neuroprotective drugs include injections, lyophilized powder injections, and oral preparations.
6. The application of daylily lactone A according to claim 5, characterized in that... The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.
7. The application of daylily lactone A according to claim 4, characterized in that... The daylily lactone A, as an active ingredient in neuroprotective drugs, is used to improve the survival rate of H2O2-induced HT22 cell damage.