Hemerocallis fulva lactone A as well as preparation method and application thereof

Abstract

The invention discloses hemerocallis fulva lactone A as well as a preparation method and application thereof, and relates to a substance with in-vitro neuroprotective activity as well as a preparation method and application thereof. The invention provides a novel substance with in-vitro neuroprotective activity and a preparation method and application thereof. The molecular formula of the day lily flower lactone A is C17H28O5. The method comprises the following steps: 1, extracting hemerocallis fulva by using an ethanol water solvent with the volume concentration of 80%; 2, carrying out rotary evaporation to remove ethanol, dispersing the concentrated solution with water, and loading the dispersed concentrated solution on a D101 macroporous resin column; and 3, carrying out separation and purification on the eluted fraction by using ethanol with the macroporous resin volume concentration of 50% through silica gel column chromatography, gel column chromatography and preparative high performance liquid chromatography to obtain the day lily lactone A. Hemerocallis fulva lactone A is used for preparing neuroprotective drugs. The hemerocallis fulva lactone A can improve the survival rate of H2O2 induced HT22 cell injury, and when the concentration of the hemerocallis fulva lactone A is 50 [mu] mol / L, the survival rate of H2O2 induced HT22 cell injury is improved from 63.77% to 81.46%, which shows that the hemerocallis fulva lactone A has good in-vitro neuroprotective activity. The invention belongs to the field of research on effective components of natural medicines.

Description

Technical Field

[0001] The present invention relates to a novel substance with in vitro neuroprotective activity and a method and application thereof. Background Art

[0002] Daylily (also known as day lily, day lily) is the dried flower bud of Hemerocallis citrina Baroni, a plant of the genus Hemerocallis in the Liliaceae family. Daylily is included in the Compendium of Materia Medica. It is cool in nature, sweet in taste, and non-toxic. It has the effects of removing dampness and heat, benefiting the chest and diaphragm, calming the five internal organs, making people happy, and improving eyesight. It can be used to treat red and astringent urine, body heat, and alcohol jaundice. Modern pharmacological studies have shown that it has pharmacological effects such as anti-depression, anti-inflammatory, antioxidant, and neuroprotective.

[0003] Modern pharmaceutical research shows that the main active ingredients of daylilies are flavonoids, phenolic acids, fatty acids, anthraquinones, dihydrostilbenes, etc., which have antidepressant, neuroprotective, anti-inflammatory, antioxidant and other effects, with little toxic side effects, and are very valuable sources of plant neuroprotective drugs.

[0004] Daylily flowers as food have geographical indication product standards such as Qidong, Dali, and Huaiyang. Daylily flowers are a long-used medicinal plant that is both a medicine and a food. In order to improve their quality standards and discover more indicator components with neuroprotective activity, it is of great significance to systematically chemically separate them.

[0005] Oxidative stress refers to a pathological state in which the balance between the oxidative and antioxidant systems in the body is broken when the body is subjected to various harmful stimuli, and free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are overproduced, exceeding the clearance capacity of the cell's antioxidant defense mechanism, thus leading to cell and tissue damage. Neuroprotection refers to a strategy to prevent, delay or alleviate nerve cell damage and prevent neurological degeneration through various means (such as drug intervention, lifestyle adjustment, physical therapy, etc.). Its purpose is to maintain the normal structure and function of the nervous system, especially for those neuronal groups that are vulnerable to damage, such as hippocampal neurons in the brain (closely related to learning and memory) and substantia nigra neurons (vulnerable 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 key role in neural functions such as learning and memory. By using the H2O2-induced HT22 cell injury model, we can screen out compounds that can effectively protect cells from damage, and further conduct in-depth pharmacological and toxicological studies on them. It is expected that new neuroprotective drugs will be developed to provide more drug options for the clinical treatment of neurological diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a new compound with in vitro neuroprotective activity and a preparation method and application thereof.

[0007] Hemerocallis lactone A, wherein the molecular formula of the hemerocallis lactone A is C 17 H 28 O5, molecular structure formula is:

[0008]

[0009] The preparation method of the hemerocallis lactone A is as follows:

[0010] 1. Place the daylily flowers in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold soaking extraction with ethanol having a volume concentration of 80%, extracting for 72 hours each time, 3 times, and combining to obtain an extract;

[0011] 2. Concentrate the extract obtained in step 1 until it has no alcohol taste, dilute it with water and apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with a volume concentration of 15%, 30%, 50%, 70%, and 90% in sequence;

[0012] 3. The eluted fraction with a volume concentration of 50% in step 2 is separated by silica gel column chromatography, and eluted in sequence with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 is applied to hydroxypropyl dextran gel column chromatography, using methanol as an eluent, and eluting at a speed of 1.5 mL / min, with each 15 mL being an eluted fraction. When the color band drops above the column outlet, the fraction is taken, and the samples of the 16th to 18th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 22.5 min in the high performance liquid chromatography is collected to obtain daylily lactone A.

[0013] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction, and the extraction temperature is room temperature.

[0014] The preparative HPLC in step 3 is a Waters 2535 semi-preparative HPLC with a Waters 2998 DAD detector as the detector and a C 18 Reverse phase silica gel was used as the filler of the chromatographic column, and a mixture of acetonitrile and water in a volume ratio of 42:58 was used as the mobile phase, with a flow rate of 3 ml / min.

[0015] The hemerocallis lactone A is used for preparing 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 hemerocallis lactone A is used as an active ingredient of neuroprotective drugs to improve the survival rate of HT22 cells damaged by H2O2.

[0019] The chemical name of the hemerocallis lactone A of the present invention is 11-(2-hydroxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl)undecanoic acid, and the molecular formula is C 17 H 28 O5.

[0020] The hemerocallis lactone A 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 daylily flowers 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 ethanol extraction, macroporous adsorption resin, silica gel column chromatography, hydroxypropyl dextran gel column chromatography and preparative liquid chromatography. The operation method is simple and rapid, and the compound separated by the method has a high purity. In addition, studies have shown that the above compound has neuroprotective activity.

[0024] The new compound can improve the survival rate of HT22 cells damaged by H2O2. When the compound is at a concentration of 50 μM, 25 μM, 12.5 μM, and 0 μM, the survival rates of HT22 cells damaged by H2O2 are 81.46%, 75.60%, 71.16%, and 63.77%, respectively. This indicates that it has good in vitro neuroprotective activity. The hemerocallis lactone A of the present invention can be prepared into neuroprotective drugs such as injections, lyophilized powder injections, infusions, or oral preparations (including tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations). DETAILED DESCRIPTION

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

[0026] Specific implementation method 1: In this implementation method, the hemerocallis lactone A has a molecular formula of C 17 H 28 O5, molecular structure formula is:

[0027]

[0028] Specific implementation method 2: The preparation method of daylily lactone A described in specific implementation method 1 is as follows:

[0029] 1. Place the daylily flowers in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold soaking extraction with ethanol having a volume concentration of 80%, extracting for 72 hours each time, 3 times, and combining to obtain an extract;

[0030] 2. Concentrate the extract obtained in step 1 until it has no alcohol taste, dilute it with water and apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with a volume concentration of 15%, 30%, 50%, 70%, and 90% in sequence;

[0031] 3. The eluted fraction with a volume concentration of 50% in step 2 is separated by silica gel column chromatography, and eluted in sequence with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 is applied to hydroxypropyl dextran gel column chromatography, using methanol as an eluent, and eluting at a speed of 1.5 mL / min, with each 15 mL being an eluted fraction. When the color band drops above the column outlet, the fraction is taken, and the samples of the 16th to 18th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 22.5 min in the high performance liquid chromatography is collected to obtain daylily lactone A.

[0032] Specific implementation method three: This implementation method is different from specific implementation method two in that the pressure of the Soxhlet dynamic extraction and concentration unit in step one is set to normal pressure extraction, and the extraction temperature is normal temperature. The rest is the same as specific implementation method two.

[0033] Specific embodiment 4: This embodiment is different from specific embodiment 2 or 3 in that the preparative high performance liquid chromatograph in step 3 is a Waters 2535 semi-preparative high performance liquid chromatograph, a Waters 2998 DAD detector is used as the detector, and a C 18 Reverse phase silica gel was used as the filler of the chromatographic column, and a mixture of acetonitrile and water in a volume ratio of 42:58 was used as the mobile phase, with a flow rate of 3 ml / min. Others were the same as those in the second or third embodiment.

[0034] Specific embodiment 5: The hemerocallis lactone A described in specific embodiments 1 to 4 is used for preparing neuroprotective drugs.

[0035] Specific embodiment 6: This embodiment is different from specific embodiment 5 in that the dosage form of the neuroprotective drug includes injection, lyophilized powder injection, and oral preparation. The rest is the same as specific embodiment 5.

[0036] Specific embodiment 7: This embodiment is different from specific embodiment 6 in that the oral preparation includes tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations. The rest is the same as specific embodiment 6.

[0037] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that the hemerocallis lactone A is used as an active ingredient of a neuroprotective drug to improve the survival rate of HT22 cells damaged by H2O2. The rest is the same as specific embodiments one to seven.

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

[0039] Experiment 1:

[0040] Hemerocallis lactone A, wherein the molecular formula of the hemerocallis lactone A is C 17 H 28 O5, molecular structure formula is:

[0041]

[0042] The preparation method of the hemerocallis lactone A is as follows:

[0043] 1. Place the daylily flowers in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold soaking extraction with ethanol having a volume concentration of 80%, extracting for 72 hours each time, 3 times, and combining to obtain an extract;

[0044] 2. Concentrate the extract obtained in step 1 until it has no alcohol taste, dilute it with water and apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with a volume concentration of 15%, 30%, 50%, 70%, and 90% in sequence;

[0045] 3. The eluted fraction with a volume concentration of 50% in step 2 is separated by silica gel column chromatography, and eluted in sequence with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 is applied to hydroxypropyl dextran gel column chromatography, using methanol as an eluent, and eluting at a speed of 1.5 mL / min, with each 15 mL being an eluted fraction. When the color band drops above the column outlet, the fraction is taken, and the samples of the 16th to 18th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 22.5 min in the high performance liquid chromatography is collected to obtain daylily lactone A.

[0046] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction, and the extraction temperature is room temperature.

[0047] The preparative HPLC in step 3 is a Waters 2535 semi-preparative HPLC with a Waters 2998 DAD detector as the detector and a C 18 Reverse phase silica gel was used as the filler of the chromatographic column, and a mixture of acetonitrile and water in a volume ratio of 42:58 was used as the mobile phase, with a flow rate of 3 ml / min.

[0048] This experiment used 10.0 kg of dried daylily flowers, and finally obtained 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 according to 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 Hemerocallis lactone A of the present invention

[0050]

[0051] Experiment 2:

[0052] The hemerocallis lactone A prepared in Experiment 1 was used to increase the survival rate of HT22 cells damaged by H2O2, and the steps were as follows:

[0053] 1. Drug preparation: Dissolve Hemerocallis lactone A in DMSO to prepare a 50mmol / L stock solution. Dilute with PBS to test concentrations of 50μmol / L, 25μmol / L, and 12.5μmol / L.

[0054] 2. Cell culture

[0055] Mouse hippocampal neuron HT22 cells were grown in DMEM culture 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.

[0056] 3. CCK-8 test of the effect of hemerocallis lactone A on cell proliferation

[0057] Mouse hippocampal neuronal cells HT22 cells were cultured at 2×10 4The cells were inoculated at a density of 100 μL / well in a 96-well plate. After the cells adhered to the wall, they were treated with different concentrations of daylily lactone A for 24 hours. The supernatant was removed and discarded. 100 μL of complete culture medium solution containing 10% CCK-8 was added to each well. After the 96-well plate was incubated in an incubator for 30 minutes, the absorbance (OD value) at 450 nm was measured with an enzyme marker. The wells with only CCK-8 solution and complete culture medium were recorded as complete blank groups, and all groups had three replicates. In this way, the safe concentration of daylily lactone A was determined. The results showed that daylily lactone A had no killing ability on cells at the tested concentration.

[0058] 4. CCK-8 test of the protective effect of hemerocallis lactone A on H2O2-induced cell damage model

[0059] HT22 cells with good growth status were inoculated in a sterile 96-well plate at a density of 2×10 4 / well, complete medium 100μL / well, mix well and place in a cell culture incubator for overnight culture. When the cells grow to 70-80% of the bottom area of ​​the 96-well plate, take out the 96-well plate, use a pipette to remove the supernatant culture medium, add 100μL of cell culture medium to the blank control group and model group, and add cell culture medium containing 50μmol / L, 25μmol / L, and 12.5μmol / L hemerocallis lactone A to the drug-treated group, respectively. Set up three replicate wells for each group and place in a cell culture incubator for continued culture. After 24h, aspirate the cell supernatant, add complete medium containing 50μmol / L H2O2 to each well except the blank control well, mix well and place in a cell culture incubator for continued culture for 2h. After the action was over, 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 50μmol / L, 25μmol / L, and 12.5μmol / L, the survival rates of HT22 cells induced by H2O2 were 81.46%, 75.60%, and 71.16%, respectively, while the cell survival rate of the model group without drug addition was 63.77%, indicating that Hemerocallis lactone A has good in vitro neuroprotective activity.

Claims

1. Hemerocallis lactone A, characterized in that The molecular formula of the hemerocallis lactone A is C 17 H 28 O5, molecular structure formula is:

2. The method for preparing the hemerocallis lactone A according to claim 1, characterized in that The preparation method of the hemerocallis lactone A is as follows:

1. Place the daylily flowers in an extraction tank of a Soxhlet dynamic extraction and concentration unit, and perform cold soaking extraction with ethanol having a volume concentration of 80%, extracting for 72 hours each time, 3 times, and combining to obtain an extract; 2. Concentrate the extract obtained in step 1 until it has no alcohol taste, dilute it with water and apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with a volume concentration of 15%, 30%, 50%, 70%, and 90% in sequence; 3. The eluted fraction with a volume concentration of 50% in step 2 is separated by silica gel column chromatography, and eluted in sequence with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:2, 100:5, 100:10, 100:20, 100:50, and 100:100, and the eluate with a volume ratio of dichloromethane to methanol of 100:20 is applied to hydroxypropyl dextran gel column chromatography, using methanol as an eluent, and eluting at a speed of 1.5 mL / min, with each 15 mL being an eluted fraction. When the color band drops above the column outlet, the fraction is taken, and the samples of the 16th to 18th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 22.5 min in the high performance liquid chromatography is collected to obtain daylily lactone A.

3. The method for preparing hemerocallis lactone A according to claim 2, characterized in that The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to normal pressure extraction, and the extraction temperature is room temperature.

4. The method for preparing hemerocallis lactone A according to claim 2, characterized in that The preparative HPLC in step 3 was a Waters 2535 semi-preparative HPLC with a Waters 2998 DAD detector as the detector and a C 18 Reverse phase silica gel was used as the filler of the chromatographic column, and a mixture of acetonitrile and water in a volume ratio of 42:58 was used as the mobile phase, with a flow rate of 3 ml / min.

5. The use of hemerocallis lactone A according to claim 1, characterized in that The hemerocallis lactone A is used for preparing neuroprotective drugs.

6. The use of hemerocallis lactone A according to claim 5, characterized in that The dosage forms of the neuroprotective drugs include injections, lyophilized powder injections, and oral preparations.

7. The use of hemerocallis lactone A according to claim 6, characterized in that The oral preparations include tablets, granules, soft capsules, hard capsules, oral liquids, and sustained-release preparations.

8. The use of hemerocallis lactone A according to claim 5, characterized in that The hemerocallis lactone A is used as an active ingredient of neuroprotective drugs to improve the survival rate of HT22 cells damaged by H2O2.

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