Hemerocallis fulva glycoside A as well as preparation method and application thereof
By extracting and purifying the hemiliaceae A from the hemiliaceae flower, the problem of overproduction of NO caused by excessive activation of BV2 cells was solved, and the effect of significantly inhibiting NO release was achieved, demonstrating its anti-inflammatory effect in central nervous system inflammation.
Patent Information
- Application Number
- CN202411878386.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-25
- Estimated Expiration
- 2044-12-19
AI Technical Summary
During the inflammation of the central nervous system, excessive activation of BV2 cells leads to excessive production of NO, triggering cytotoxicity and vicious cycles, and aggravating nervous system damage. It is difficult for the existing technology to effectively inhibit this process.
A new compound, Hemiliaceae A, was extracted from Historicum flower, and purified by 80% ethanol extraction, macroporous resin column elution, silica gel column chromatography and hydroxypropyl dextran gel column chromatography, and prepared into an anti-inflammatory drug.
Hemerocalin A can significantly inhibit the NO release of BV2 cells induced by LPS, with inhibition rates of 84.9%, 68.7%, 45.2%, 31.2%, and 12.6% respectively. At different concentrations, it showed good in vitro anti-inflammatory activity.
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Abstract
Description
Technical Field
[0001] The present invention relates to a new substance with in vitro anti-inflammatory effect 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, and antioxidant.
[0003] Modern pharmaceutical research shows that the main active ingredients of daylilies are flavonoids, phenolic acids, anthraquinones, dihydrostilbenes, etc., most of which have anti-inflammatory and antioxidant effects, with small toxic and side effects, and are very valuable sources of plant anti-inflammatory drugs.
[0004] After the daylily root was included in the 1977 edition of the Chinese Pharmacopoeia, daylily has not been included in any edition of the pharmacopoeia. Daylily has geographical indication product standards in Qidong, Dali, Huaiyang, etc. As a long-used medicinal plant that is both a medicine and a food, it is of great significance to systematically chemically separate daylily flowers in order to improve their quality standards and discover more index components with anti-inflammatory activity.
[0005] In the inflammatory process of the central nervous system, BV2 cells, as representatives of microglia, play a similar role to in situ microglia in vivo. Under normal physiological conditions, BV2 cells are in a quiescent state, playing a role in monitoring the surrounding microenvironment and maintaining neuronal homeostasis. However, when exposed to pathological stimuli such as pathogen infection, brain damage, and neurotoxins, BV2 cells are rapidly activated and transformed into a cell morphology with immune activity. For example, under the stimulation of lipopolysaccharide (LPS), BV2 cells express a variety of inflammatory-related factors after activation. If BV2 cells are overactivated and continuously release a large number of inflammatory factors, it may cause the so-called "inflammatory storm", leading to neuronal damage, affecting normal physiological functions such as nerve conduction, and then participating in pathological processes such as neurodegenerative diseases (the pathogenesis of diseases such as Alzheimer's disease and Parkinson's disease involves abnormal activation of microglia including BV2 cells and dysregulated inflammatory responses). When BV2 cells are stimulated by bacterial lipopolysaccharide (LPS), the intracellular signaling pathway is activated, which upregulates the expression of iNOS and synthesizes a large amount of NO. Excessive production of high concentrations of NO by BV2 cells will show cytotoxic effects. On the one hand, high concentrations of NO can react quickly with superoxide anions to generate peroxynitrite, a substance with strong oxidizing properties that can damage biological macromolecules such as proteins, lipids, and DNA in cells, leading to cell dysfunction or even death; on the other hand, excessive NO can also further aggravate the inflammatory response by affecting some signal transduction pathways in cells, forming a vicious cycle and exacerbating damage to the central nervous system. 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] Hemerocallis glycoside A, wherein the molecular formula of the hemerocallis glycoside A is C 23 H 28 O 10 , the molecular structure is:
[0008]
[0009] The preparation method of the hemerocallis glycoside 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 then apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with volume concentrations 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 with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200 in sequence, and the eluate with a volume ratio of petroleum ether and ethyl acetate of 100:100 is applied to hydroxypropyl dextran gel column chromatography, using methanol as 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 collected, and the samples of the 35th-36th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 7.8 min in the high performance liquid chromatography is collected to obtain hemerocallis glycoside A.
[0013] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to extraction at normal temperature and pressure.
[0014] The high performance liquid chromatograph in step 3 is a Waters 2535 semi-preparative high performance liquid chromatograph, 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 the mobile phase was a mixture of acetonitrile and water in a volume ratio of 38:62, with a flow rate of 3 ml / min.
[0015] The hemerocallis glycoside A 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 hemerocallis glycoside A is used as an active ingredient of an anti-inflammatory drug to inhibit the NO release of microglial cells BV2 induced by LPS.
[0019] The chemical name of the hemerocallis glycoside A of the present invention is [3-methyl-2-hydroxy-6-(4-hydroxyphenethyl)-methyl benzoate]-4-O-β-D-glucopyranoside, and the molecular formula is C 23 H 28 O 10 .
[0020] The compound prepared by 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 invention provides a new compound derived from daylily flowers and a method for extracting, separating and purifying the new compound of the invention. The new compound is successfully obtained by using 80% ethanol extraction, macroporous adsorption resin, silica gel column chromatography, hydroxypropyl dextran gel column chromatography and a preparative liquid phase method for separation and purification. The operation method is simple and rapid, and the compound separated by the method has a high purity and has anti-inflammatory activity.
[0024] The new compound can inhibit the increase of NO content caused by LPS-induced mouse microglial BV2 cells. When the compound is at a concentration of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, the inhibition rate of NO production is 84.9%, 68.7%, 45.2%, 31.2%, and 12.6%, respectively. It shows that it has good in vitro anti-inflammatory activity. The hemerocallis glycoside A of the present invention can be made into an anti-inflammatory drug in the form of injection, lyophilized powder injection, infusion or oral preparation (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 glycoside A has a molecular formula of C 23 H 28 O 10 , the molecular structure is:
[0027]
[0028] Specific implementation method 2: The preparation method of hemerocallis glycoside 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 then apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with volume concentrations 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 with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200 in sequence, and the eluate with a volume ratio of petroleum ether and ethyl acetate of 100:100 is applied to hydroxypropyl dextran gel column chromatography, using methanol as 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 collected, and the samples of the 35th-36th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 7.8 min in the high performance liquid chromatography is collected to obtain hemerocallis glycoside 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 high performance liquid chromatograph in step 3 is a Waters 2535 semi-preparative high performance liquid chromatograph, with a Waters 2998 DAD detector as the detector and a C 18 Reverse phase silica gel is used as the filler of the chromatographic column, the mobile phase is a mixture of acetonitrile and water in a volume ratio of 38:62, and the flow rate is 3 ml / min. Others are the same as those in the second or third embodiment.
[0034] Specific implementation method 5: The hemerocallis glycoside A described in specific implementation methods 1 to 4 is used to prepare 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 one to seven in that the hemerocallis glycoside A is used as an active ingredient of an anti-inflammatory drug to inhibit the NO release of microglial cells BV2 induced by LPS. 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 glycoside A, wherein the molecular formula of the hemerocallis glycoside A is C 23 H 28 O 10 , the molecular structure is:
[0041]
[0042] The preparation method of the hemerocallis glycoside 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 then apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with volume concentrations 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 with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200 in sequence, and the eluate with a volume ratio of petroleum ether and ethyl acetate of 100:100 is applied to hydroxypropyl dextran gel column chromatography, using methanol as 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 collected, and the samples of the 35th-36th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 7.8 min in the high performance liquid chromatography is collected to obtain hemerocallis glycoside A.
[0046] The pressure of the Soxhlet dynamic extraction and concentration unit in step 1 is set to extraction at normal temperature and pressure.
[0047] The high performance liquid chromatograph in step 3 is a Waters 2535 semi-preparative high performance liquid chromatograph, 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 the mobile phase was a mixture of acetonitrile and water in a volume ratio of 38:62, with a flow rate of 3 ml / min.
[0048] This experiment used 10.0 kg of dried daylily flowers, and finally obtained 6.85 mg of daylily glycoside A. The new compound was named [3-methyl-2-hydroxy-6-(4-hydroxyphenethyl)-methyl benzoate]-4-O-β-D-glucopyranoside according to its structure. Table 1 shows the NMR data of the new compound: 1 H-NMR and 13 C-NMR in CD 3 In OD.
[0049] Table 1: NMR data of Hemerocallis glycoside A of the present invention
[0050]
[0051]
[0052] Experiment 2:
[0053] The inhibitory activity of Hemerocallis glycoside A prepared in Experiment 1 on LPS-induced NO production in BV2 cells was tested in the following steps:
[0054] 1. Drug preparation: Dissolve Hemerocallis glycoside A in DMSO to prepare a 50mmol / L stock solution. Dilute with PBS to test concentrations of 50μmol / L, 25μmol / L, 12.5μmol / L, 6.25μmol / L, and 3.125μmol / L.
[0055] 2. Cell culture
[0056] Mouse microglial BV2 cells were grown in DMEM containing 10% fetal bovine serum and in a 5% CO 2 The cells were suspended and cultured in an incubator at 37°C and saturated humidity, and the cells in the logarithmic growth phase were used for the experiment.
[0057] 3. CCK-8 test of the effect of drugs on cell proliferation
[0058] BV2 cells were collected at 3×10 4 The 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 Hemerocallis glycoside 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 Hemerocallis glycoside A was determined. The results showed that Hemerocallis glycoside A had no killing ability on cells at the tested concentration.
[0059] 4. Griess colorimetric method to test the inhibitory activity of drugs on NO production
[0060] BV2 cells with good growth status were inoculated in a sterile 96-well plate at a density of 3×10 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 fluid to the blank control group and model group, and add cell culture fluid containing monomer compounds at concentrations of 3.125, 6.250, 12.50, 25.00, and 50.00μM, respectively, to the drug-treated group. Set up three replicate wells for each group and place in a cell culture incubator for continued culture. After 4h, LPS was added to each well except the blank control well, so that the concentration of LPS in the culture medium was 1μg / mL, and after mixing, it was placed in a cell culture incubator for continued culture for 24h. After the drug effect ends, take 50μL of the cell supernatant from each well and transfer it to another sterile 96-well plate. 50 μL of Griess Reagent I and Griess Reagent II were added to each well in a dark place, and the absorbance at 540 nm was measured with an ELISA reader. The inhibition rate of each compound in inhibiting NO release was calculated, inhibition rate = (OD 模型组 -OD 给药组 ) / (OD 模型组 -OD 空白对照组 ), the results showed that when the concentrations were 50μmol / L, 25μmol / L, 12.5μmol / L, 6.25μmol / L, and 3.125μmol / L, the inhibition rates were 84.9%, 68.7%, 45.2%, 31.2%, and 12.6%, respectively. IC 50 The value was 13.3 μmol / L, indicating that it has good in vitro anti-inflammatory activity.
Claims
1. Hemerocallis glycoside A, characterized in that The molecular formula of the hemerocallis glycoside A is C 23 H 28 O 10 , the molecular structure is:
2. The method for preparing hemerocallis glycoside A according to claim 1, characterized in that The preparation method of the hemerocallis glycoside 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%, each time for 72 hours, for 3 times, and combine to obtain an extract; 2. Concentrate the extract obtained in step 1 until it has no alcohol taste, dilute it with water, and then apply it to a D101 macroporous resin column, and then elute it with water and ethanol water with volume concentrations 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 with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200 in sequence, and the eluate with a volume ratio of petroleum ether and ethyl acetate of 100:100 is applied to hydroxypropyl dextran gel column chromatography, using methanol as 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 collected, and the samples of the 35th-36th fractions are combined to prepare a high performance liquid chromatography sample, and the absorption peak that appears at a retention time of 7.8 min in the high performance liquid chromatography is collected to obtain hemerocallis glycoside A.
3. The method for preparing hemerocallis glycoside 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 glycoside A according to claim 2, characterized in that The high performance liquid chromatograph in step 3 is a Waters 2535 semi-preparative high performance liquid chromatograph, 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 the mobile phase was a mixture of acetonitrile and water in a volume ratio of 38:62, with a flow rate of 3 ml / min.
5. The use of hemerocallis glycoside A according to claim 1, characterized in that The hemerocallis glycoside A is used for preparing anti-inflammatory drugs.
6. The use of hemerocallis glycoside A 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 hemerocallis glycoside 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 glycoside A according to claim 5, characterized in that The hemerocallis glycoside A is used as an active ingredient of an anti-inflammatory drug to inhibit the NO release of microglial cells BV2 induced by LPS.
Citation Information
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