Hemerocallis A, 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 present invention provides a novel substance with in vitro anti-inflammatory activity, its preparation method, and its application. The molecular formula of hemerocallicin A is C2. 23 H 28 O 10 Methods: 1. Hemerocallis flowers were extracted with 80% ethanol aqueous solvent; 2. The concentrated extract after removing ethanol by rotary evaporation was dispersed in water and loaded onto a D101 macroporous resin column; 3. The fraction was eluted with 50% ethanol from the macroporous resin, and then purified by silica gel column chromatography, gel column chromatography, and preparative high-performance liquid chromatography to obtain hemerocalin A. Hemerocalin A is used to prepare anti-inflammatory drugs. The IC50 of hemerocalin A in this invention inhibited the release of the pro-inflammatory factor NO from LPS-stimulated mouse microglia (BV2) by [the method described in the original text]. 50 The value was 13.3 μmol / L, 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 method, and its 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, and antioxidant pharmacological effects.

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

[0004] After the root of the daylily was included in the 1977 edition of the Chinese Pharmacopoeia, the daylily itself has not been included in any other editions of the pharmacopoeia. Daylily flowers, as a food product, have geographical indication product standards in Qidong, Dali, and Huaiyang. As a long-used medicinal and edible plant, the systematic chemical separation of daylily flowers is of great significance for improving its quality standards and discovering more indicative components with anti-inflammatory activity.

[0005] In the inflammatory process of the central nervous system, BV2 cells, as representatives of microglia, play a function similar to that of in situ microglia in vivo. Under normal physiological conditions, BV2 cells are in a resting state, playing a role in monitoring the surrounding microenvironment and maintaining neuronal homeostasis. However, when stimulated by pathological factors such as pathogen infection, brain injury, or neurotoxins, BV2 cells are rapidly activated and transform into an immune-active cell morphology. For example, under the stimulation of lipopolysaccharide (LPS), activated BV2 cells express a variety of inflammation-related factors. If BV2 cells are overactivated and continuously release large amounts of inflammatory factors, it may cause a so-called "cytokine storm," leading to neuronal damage, affecting normal physiological functions such as nerve conduction, and thus participating in pathological processes such as neurodegenerative diseases (the pathogenesis of diseases such as Alzheimer's disease and Parkinson's disease involves abnormal activation and dysregulation of microglia, including BV2 cells). When BV2 cells are stimulated by bacterial lipopolysaccharide (LPS), intracellular signaling pathways are activated, resulting in upregulation of iNOS expression and the synthesis of large amounts of NO. Excessive production of high concentrations of NO by BV2 cells can exhibit cytotoxic effects. On one hand, high concentrations of NO can rapidly react with superoxide anions to produce peroxynitrite, a highly oxidizing substance that damages intracellular macromolecules such as proteins, lipids, and DNA, leading to cell dysfunction and even death. On the other hand, excessive NO can also further exacerbate the inflammatory response by affecting intracellular signal transduction pathways, creating a vicious cycle and exacerbating damage to the central nervous system. Therefore, substances that can inhibit inflammatory responses are of great significance for protecting public health and improving people's 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] Hemerocallis glycoside A, wherein the molecular formula of hemerocallis glycoside A is C 23 H 28 O 10 The molecular structural formula is:

[0008]

[0009] The preparation method of the daylily glycoside 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 (v / v) 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 it with water, and then load it onto a D101 macroporous resin column. Then, elute it sequentially with water and ethanol water with volume concentrations of 15%, 30%, 50%, 70%, and 90%.

[0012] 3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using a mixed solvent of petroleum ether and ethyl acetate at volume ratios of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200. Then, use a 100:100 volume ratio of petroleum ether and ethyl acetate eluent for hydroxypropyl dextran gel chromatography, 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 above the column outlet. Combine the 35th and 36th fractions to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak appearing at retention time 7.8 min in the HPLC, which yields hemerocallin A.

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

[0014] Step 3 describes a Waters 2535 semi-preparative HPLC system, using a Waters 2998 DAD detector and a C000S ... 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water with a volume ratio of 38:62, with a flow rate of 3 ml / min.

[0015] The hemerocallis glycoside A 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 daylily glycoside A, as the active ingredient of the anti-inflammatory drug, is used to inhibit the release of NO from LPS-induced microglia BV2.

[0019] The chemical name of the hemerocallis glycoside A of this invention is [3-methyl-2-hydroxy-6-(4-hydroxyphenethyl)-methyl benzoate]-4-O-β-D-glucopyranoside, and its molecular formula is C2. 23 H 28 O 10 .

[0020] The compounds prepared by 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 new compound derived from daylily flowers and a method for extracting, separating, and purifying the new compound. The method employs 80% 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 new 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 can inhibit the increase in NO levels induced by LPS in mouse microglia (BV2) cells. At concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, the compound exhibited inhibition rates of 84.9%, 68.7%, 45.2%, 31.2%, and 12.6% on NO production, respectively, indicating good in vitro anti-inflammatory activity. The hemerocallicin 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 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 hemerocallis glycoside A, the molecular formula of which is C. 23 H 28 O 10 The molecular structural formula is:

[0027]

[0028] Specific Implementation Method Two: The preparation method of daylily glycoside 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 (v / v) 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 it with water, and then load it onto a D101 macroporous resin column. Then, elute it sequentially with water and ethanol water with volume concentrations of 15%, 30%, 50%, 70%, and 90%.

[0031] 3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using a mixed solvent of petroleum ether and ethyl acetate at volume ratios of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200. Then, use a 100:100 volume ratio of petroleum ether and ethyl acetate eluent for hydroxypropyl dextran gel chromatography, 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 above the column outlet. Combine the 35th and 36th fractions to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak appearing at retention time 7.8 min in the HPLC, which yields hemerocallin 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 chromatograph mentioned in step three is a Waters 2535 semi-preparative high-performance liquid chromatograph, using a Waters 2998 DAD detector as the detector, and C... 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 38:62, 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 glycoside A described in Specific Implementation Methods 1 to 4 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 One to Seven in that the hemerocalcin A is used as the active ingredient of an anti-inflammatory drug to inhibit the release of NO from LPS-induced microglia BV2. 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 glycoside A, wherein the molecular formula of hemerocallis glycoside A is C 23 H 28 O 10 The molecular structural formula is:

[0041]

[0042] The preparation method of the daylily glycoside 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 (v / v) 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 it with water, and then load it onto a D101 macroporous resin column. Then, elute it sequentially with water and ethanol water with volume concentrations of 15%, 30%, 50%, 70%, and 90%.

[0045] 3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using a mixed solvent of petroleum ether and ethyl acetate at volume ratios of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:200. Then, use a 100:100 volume ratio of petroleum ether and ethyl acetate eluent for hydroxypropyl dextran gel chromatography, 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 above the column outlet. Combine the 35th and 36th fractions to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak appearing at retention time 7.8 min in the HPLC, which yields hemerocallin A.

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

[0047] Step 3 describes a Waters 2535 semi-preparative HPLC system, using a Waters 2998 DAD detector and a C000S ... 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water with 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, ultimately obtaining 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 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 hemerocallinoside A in this invention

[0050]

[0051]

[0052] Experiment 2:

[0053] The inhibitory activity of hemerocallicin A prepared in Experiment 1 on LPS-induced NO production in BV2 cells was investigated, and the steps are as follows:

[0054] 1. Drug preparation: Hemerocallis glycoside 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, 12.5 μmol / L, 6.25 μmol / L, and 3.125 μmol / L.

[0055] 2. Cell Culture

[0056] Mouse microglia BV2 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.

[0057] 3. Effects of CCK-8 assay on cell proliferation

[0058] BV2 cells at 3×10 4 Cells were seeded at a density of [number] cells / well in 96-well plates. After adhesion, the cells were treated with different concentrations of hesperidin A for 24 hours. The plates were then removed, the supernatant discarded, and 100 μL of 10% CCK-8 complete culture medium solution was added to each well. The 96-well plates were incubated for 30 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 hesperidin A. The results showed that hesperidin A had no cell-killing ability at the tested concentration.

[0059] 4. Griess colorimetric method for testing the inhibitory activity of drugs on NO production.

[0060] Healthy BV2 cells were seeded into sterile 96-well plates at a density of 3 × 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 monomeric compounds at concentrations of 3.125, 6.250, 12.50, 25.00, and 50.00 μM were added to the drug-treated groups, respectively. Three replicates were set up for each group, and the plates were incubated for further culture. After 4 hours, LPS was added to each well except for the blank control wells to bring the LPS concentration in the culture medium to 1 μg / mL. The plates were mixed and incubated for another 24 hours. After the drug effect was complete, 50 μL of cell supernatant from each well was transferred to another sterile 96-well plate. Add 50 μL of Griess Reagent I and Griess Reagent II sequentially to each well in the dark, and measure the absorbance at 540 nm using a microplate reader. Calculate the inhibition rate of NO release for each compound, where inhibition rate = (OD200) / (NO200) * ... 模型组 -OD 给药组 ) / (OD 模型组 -OD 空白对照组 The results showed that the inhibition rates were 84.9%, 68.7%, 45.2%, 31.2%, and 12.6% at concentrations of 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, and 3.125 μmol / L, respectively. The IC50 was calculated using SPSS software. 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 daylily glycoside A is C 23 H 28 O 10 The molecular structural formula is: 。 2. The method for preparing hemerocallis glycoside A according to claim 1, characterized in that... The preparation method of the daylily glycoside 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 (v / v) 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 it with water, and then load it onto a D101 macroporous resin column. Then, elute it sequentially with water and ethanol water with volume concentrations of 15%, 30%, 50%, 70%, and 90%.

3. Separate the 50% volume concentration eluent from step 2 by silica gel column chromatography, using a mixed solvent of petroleum ether and ethyl acetate at volume ratios of 100:4, 100:10, 100:20, 100:40, 100:100, and 100:

200. Then, use a 100:100 volume ratio of petroleum ether and ethyl acetate eluent for hydroxypropyl dextran gel chromatography, 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 above the column outlet. Combine the 35th and 36th fractions to prepare a high-performance liquid chromatography (HPLC) sample. Collect the absorption peak appearing at retention time 7.8 min in the HPLC, which yields hemerocallin A.

3. The method for preparing daylily glycoside A according to claim 2, characterized in that... The Soxhlet dynamic extraction and concentration unit described in step one is set to atmospheric pressure extraction and ambient temperature extraction.

4. The method for preparing hemerocallis glycoside A according to claim 2, characterized in that... Step 3 describes a Waters 2535 semi-preparative HPLC system, using a Waters 2998 DAD detector and a C000S ... 18 The reversed-phase silica gel column was used as the packing material, and the mobile phase was a mixture of acetonitrile and water with a volume ratio of 38:62, with a flow rate of 3 ml / min.

5. The application of the daylily glycoside A according to claim 1, characterized in that... The hemerocallis glycoside A is used to prepare anti-inflammatory drugs.

6. The application of daylily glycoside A 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 daylily 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 application of daylily glycoside A according to claim 5, characterized in that... The daylily glycoside A, as the active ingredient of the anti-inflammatory drug, is used to inhibit the release of NO from LPS-induced microglia BV2.

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