Flavone glycoside compound and application thereof in preparation of anti-inflammatory drugs

Abstract

The application discloses a flavone glycoside compound and application thereof in preparation of anti-inflammatory drugs, and the application comprises the following steps: step one, alcohol extraction of raw materials; step two, resin column chromatography; step three, silica gel column chromatography; step four, polyamide column chromatography; step five, ODS column chromatography; step six, obtaining of the target compound; and step seven, application of the target compound; in the step one, the alcohol extraction process specifically comprises: 2-4 times of heating reflux extraction, each time for 1-3 hours; in the step six, the separation method is HPLC high performance liquid chromatography; the flavone carbon glycoside compound with anti-inflammatory activity is obtained through the following steps of solvent extraction, macroporous adsorption resin column chromatography, silica gel column chromatography, polyamide column chromatography, ODS column chromatography and preparation liquid phase separation by using dried flowers of the Trollius chinensis G.Lodd, and the application has the advantages of simple separation method and easy-to-obtain raw materials, and the obtained compound is expected to be developed into an anti-inflammatory lead compound, and has important significance for development of anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a flavonoid glycoside compound and its application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation is a crucial immune response, a way for the body to heal itself after injury, repair damaged tissue, and fight off pathogens. Inflammation is a complex process involving the body's white blood cells and the chemicals they produce, such as antibodies and cytokines, which are released into the bloodstream or affected tissues to combat foreign invaders. Inflammation can be divided into acute and chronic inflammation. Acute inflammation is the body's natural defense against damaged cells, viruses, and other harmful stimuli, and usually subsides within a short period. Chronic inflammation is a systemic inflammation that lasts for months or years and is associated with almost all health problems and diseases. Chronic inflammation is a common factor contributing to many health problems, including metabolic syndrome, non-alcoholic fatty liver disease, type 2 diabetes, cancer, Alzheimer's disease, and heart disease. Therefore, researching anti-inflammatory lead compounds with novel structures is of great significance for treating inflammation. Summary of the Invention

[0003] The purpose of this invention is to provide a flavonoid glycoside compound and its application in the preparation of anti-inflammatory drugs, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a flavonoid glycoside compound, wherein the structural formula of the flavonoid glycoside compound is as follows:

[0005]

[0006] The application of a flavonoid glycoside compound in the preparation of an anti-inflammatory drug includes the following steps: Step 1, alcohol extraction of the raw material; Step 2, resin column chromatography; Step 3, silica gel column chromatography; Step 4, polyamide column chromatography; Step 5, ODS column chromatography; Step 6, obtaining the target compound; Step 7, application of the target compound.

[0007] In step one above, the dried golden lotus flower is extracted with an ethanol-water solution of 60% to 99% by volume, the soaking liquid is recovered and concentrated under reduced pressure to obtain crude ethanol extract.

[0008] In step two above, the crude ethanol extract obtained in step one is dissolved in an appropriate amount of water, centrifuged and filtered, and the filtrate is eluted by ethanol-water gradient chromatography through a macroporous resin column to obtain 5 fractions (Fr.A~Fr.E).

[0009] In step three above, the fraction Fr.C from step two is subjected to silica gel column chromatography and eluted with a gradient of dichloromethane and methanol to obtain 11 fractions (Fr.C to Fr.C11);

[0010] In step four above, the fraction Fr.C6 from step three is subjected to polyamide column chromatography and eluted with a gradient of methanol and water to obtain eight fractions (Fr.C6A to Fr.C6H).

[0011] In step five above, the fraction Fr.C6E from step four is subjected to ODS column chromatography and eluted with a methanol and water gradient to obtain five fractions (Fr.C6E1 to Fr.C6E5).

[0012] In step six above, the target flavonoid glycoside compound is obtained by separating the fraction Fr.C6E4 from step five;

[0013] In step seven above, the target flavonoid glycoside compound obtained in step six is ​​used in the preparation of anti-inflammatory drugs.

[0014] Preferably, in step one, the dried golden lotus flowers are pre-chopped.

[0015] Preferably, in step one, the alcohol extraction process specifically involves: heating and reflux extraction 2 to 4 times, each time for 1 to 3 hours.

[0016] Preferably, in step two, the volume ratio of ethanol to water is 0:1, 3:7, 5:5, 7:3, or 95:5.

[0017] Preferably, in step three, the volume ratios of dichloromethane and methanol are 98:2, 93:7, 9:1, and 0:1, respectively.

[0018] Preferably, in step four, the volume ratios of methanol and water are 1:4, 2:3, and 1:0, respectively.

[0019] Preferably, in step five, the volume ratio of methanol to water is 1:4, 1:3, 3:7, 1:1, and 1:0, respectively.

[0020] Preferably, in step six, the separation method employs HPLC (high performance liquid chromatography).

[0021] Preferably, the conditions for the HPLC high-performance liquid chromatography are: ODS Rp-18, 5 μm, 250 × 10 mm, 35% MeOH / H2O, 5 mL / min.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses dried flowers of golden lotus, and obtains flavonoid C-glycoside compounds with anti-inflammatory activity through solvent extraction, macroporous adsorption resin column chromatography, silica gel column chromatography, polyamide column chromatography, ODS column chromatography, and preparative liquid phase separation. The present invention has the advantages of simple separation method and readily available raw materials. The obtained compounds are expected to be developed into anti-inflammatory lead compounds, which is of great significance for the development of anti-inflammatory drugs. Attached Figure Description

[0023] Figure 1 The target flavonoid glycosides inhibited LPS-induced NO production in RAW264.7 macrophages (drug concentration 50 μM, action time 2 h);

[0024] Figure 2 The target flavonoid glycosides were used to inhibit the expression of the inflammation-related protein iNOS in a concentration-dependent manner (drug concentration 12.5-50 μM, action time 2 h);

[0025] Figure 3 This is a flowchart of the application method of the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Please see Figures 1-3 One embodiment of the present invention provides: a flavonoid glycoside compound, the structural formula of which is as follows:

[0028]

[0029] The application of a flavonoid glycoside compound in the preparation of an anti-inflammatory drug includes the following steps: Step 1, alcohol extraction of the raw material; Step 2, resin column chromatography; Step 3, silica gel column chromatography; Step 4, polyamide column chromatography; Step 5, ODS column chromatography; Step 6, obtaining the target compound; Step 7, application of the target compound.

[0030] In step one above, the dried golden lotus flowers after being chopped are extracted with an ethanol-water solution of 60% to 99% by volume. The extraction is carried out by heating and reflux for 2 to 4 times, each time for 1 to 3 hours. The soaking liquid is then recovered and concentrated under reduced pressure to obtain crude ethanol extract.

[0031] In step two above, the crude ethanol extract obtained in step one is dissolved in an appropriate amount of water, centrifuged and filtered, and the filtrate is eluted by ethanol-water gradient chromatography through a macroporous resin column to obtain 5 fractions (Fr.A~Fr.E) with volume ratios of ethanol to water of 0:1, 3:7, 5:5, 7:3, and 95:5, respectively.

[0032] In step three above, the fraction Fr.C from step two is subjected to silica gel column chromatography and eluted with a gradient of dichloromethane and methanol to obtain 11 fractions (Fr.C to Fr.C11), with volume ratios of dichloromethane and methanol of 98:2, 93:7, 9:1, and 0:1, respectively.

[0033] In step four above, the fraction Fr.C6 from step three is subjected to polyamide column chromatography and eluted with a methanol and water gradient to obtain eight fractions (Fr.C6A to Fr.C6H), with methanol and water volume ratios of 1:4, 2:3, and 1:0, respectively.

[0034] In step five above, the fraction Fr.C6E from step four is subjected to ODS column chromatography and eluted with a methanol and water gradient to obtain five fractions (Fr.C6E1 to Fr.C6E5), with methanol and water volume ratios of 1:4, 1:3, 3:7, 1:1, and 1:0, respectively.

[0035] In step six above, the target flavonoid glycosides are obtained by separating the fraction Fr.C6E4 from step five. The separation method is HPLC high performance liquid chromatography, and the conditions are: ODS Rp-18, 5μm, 250×10mm, 35%MeOH / H2O, 5mL / min.

[0036] In step seven above, the target flavonoid glycoside compound obtained in step six is ​​used in the preparation of anti-inflammatory drugs.

[0037] The target flavonoid glycosides obtained in the above embodiments were structurally identified as follows: the target flavonoid glycosides are yellow powders. HR-ESI-MS: m / z583.1458[M+H] + (C 29 H 27 O 13 (Calculated value 583.1446), molecular formula is C 29 H 26 O 13 Ω = 17; the target flavonoid glycosides have similar NMR data to 2″-Op-hydroxybenzoylorientin reported in the literature, and are obtained through... 1 H NMR and 13 C NMR data [δ] H3.76(3H,s)] and [δ C [56.6] It was inferred that there was one extra methoxy group, which was determined by the correlation signal δ in the HMBC spectrum. H 3.76(H-OCH3) and δ C The correlation of 162.7 (C-7) indicates that the methoxy group is attached to the parent nucleus at C-7; the derivatization reaction yields compound 2. * The sugar is in the D-configuration, and based on the terminal hydrogen J value (10.1 Hz), it was further determined to be β-D-glucose, thus establishing its structure. This leads to the identification of the target flavonoid glycoside compound. 1 H NMR and 13 The C NMR data are shown in the table below;

[0038]

[0039]

[0040] Verification of the anti-inflammatory effects of the target flavonoid glycosides:

[0041] 1. The target flavonoid glycosides inhibit LPS-induced nitric oxide production in RAW264.7 macrophages. The principle is that NO is unstable in aqueous solution and is easily oxidized to NO2. － The sample reacts with Griess Reagent I solution to yield a diazo compound (light yellow), which then undergoes a coupling reaction with Griess Reagent II solution to generate a colored compound (purple-red). The absorbance is measured at 540 nm using a microplate reader to create a standard curve, thus determining the NO content in the sample. The experimental method is as follows:

[0042] 1.1 Establishment of standard curve: The NO kit standard was diluted with DMEM culture medium containing 10% fetal bovine serum (FBS) to a final concentration of 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM. 50 μL was added to each well of a 96-well plate, with 3 replicates for each well. Then, 50 μL each of solution I and solution II were added sequentially. The plates were shaken and reacted in the dark for 10 min. The absorbance was measured at 540 nm. The concentration and OD values ​​were plotted on the x and y axes to obtain the standard curve.

[0043] 1.2 RAW264.7 macrophages were seeded into 12-well plates. After 12 hours, the cell density was observed to reach 60-70%. The positive control drug TPCA and the compound were pretreated with the cells at a final concentration of 50 μM for 2 hours. After pretreatment, LPS was added to the TPCA group, experimental group, and LPS model group to a final concentration of 1 μg / mL. The blank control group was not treated. After 24 hours, the cell supernatant was harvested for NO detection. 50 μL of the supernatant was taken, and the standard curve was established according to the above procedure. The NO content was calculated by substituting the values ​​into the standard curve.

[0044] Experimental results are as follows Figure 1 As shown, the LPS group significantly induced NO production compared to the blank control group, while the target flavonoid glycosides significantly inhibited NO production, suggesting that the target flavonoid glycosides have certain anti-inflammatory activity.

[0045] 2. Western blot analysis of the effect of target flavonoid glycosides on the expression of the inflammation-related protein iNOS. The experimental method is as follows:

[0046] 2.1 Cell Culture: After thawing the frozen HeLa cells, they were placed in DMEM medium and cultured in a cell culture incubator at 37°C with constant humidity and 5% CO2. The medium was replaced after 24 hours of culture. The cells were then passaged when the cell density reached about 70%. After 2-3 passages, cells with stable morphology and growth were obtained.

[0047] 2.2 Plating and Drug Addition: Place the cultured cells at a density of 1 × 10⁶ cells per well. 6 The concentration of the flavonoid glycosides was added to the 12-well plate, mixed thoroughly, and then placed in a cell culture incubator for culture. After the cells adhered stably, the target flavonoid glycosides were added (final concentrations of 50, 25, and 12.5 μM, respectively). At the same time, a blank control group was set up, and an equal amount of DMSO was added to it. The culture medium was then mixed thoroughly and placed in a cell culture incubator for 2 hours.

[0048] 2.3 Cell lysis: After culture, the culture medium was removed from the cells, and the cells were washed twice with pre-chilled 1×PBS solution. Then, the 12-well plate was placed on an ice box, and an appropriate amount of RIPA lysis buffer containing protease inhibitors was added to the 12-well plate. The 12-well plate was gently shaken until the lysis buffer was evenly distributed on the cell surface. After lysis for 2 min, the cells were scraped off with a cell scraper and transferred to pre-chilled 1.5 mL EP tubes. The plates were placed on an ice box and lysis continued for 30 min. The cells were vortexed for 2 s every 10 min. Finally, the lysed cells were placed in a pre-chilled 4°C centrifuge and centrifuged at 12000 rpm for 10 min. The supernatant was collected for later use.

[0049] 2.4 Determination of protein concentration: Take the supernatant from centrifugation and determine the protein concentration of the sample using the BCA method;

[0050] 2.5 Protein Gel Electrophoresis: Take the same mass of protein sample, add lysis buffer to make up the same volume, add 1 / 4 volume of 5×SDS loading buffer (containing 5% β-mercaptoethanol), incubate in a 100℃ metal bath for 5 min, and centrifuge to remove the precipitate; add an equal amount of protein sample to the comb wells of the prepared electrophoresis gel plate, controlling the total protein amount in each well to be 20-60 μg, and the volume not exceeding 30 μL; add 1 μL of protein marker to both ends, and electrophoresis at 80V; when the sample enters the separating gel, the voltage can be increased to 120V and electrophoresis can be continued until the bromophenol blue band is 1-2 cm away from the bottom of the gel.

[0051] 2.6 Transfer: After removing the gel from the gel plate, place it temporarily in the pre-cooled electrotransfer solution. Then, take a PVDF membrane of the same size (pre-treated with methanol for 1 min) and filter paper and immerse them in the electrotransfer solution. With the black side of the electrotransfer clamp facing down, place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in sequence. Remove air bubbles from the electrotransfer clamp and place them in the electrotransfer tank in the order of membranes facing the positive electrode. Perform electrotransfer in an ice box. At this time, the voltage is controlled at 90V and the current is controlled below 300mA. Electrotransfer for 60-90 min.

[0052] 2.7 Blocking: The PVDF membrane after electrotransfer was placed in TBST buffer containing 5% skim milk and blocked on a horizontal shaker at room temperature for 1 hour;

[0053] 2.8 Primary antibody reaction: After removing the PVDF membrane from the blocking solution, cut the required protein bands, wash them twice with an appropriate amount of TBST buffer, place them in an antibody incubation box, add the corresponding primary antibodies, and dilute all primary antibodies with 5% BSA 1:1000 (anti-β-actin, anti-iNOS); then incubate the PVDF membrane overnight at 4°C with shaking.

[0054] 2.9 Secondary antibody reaction: After recovering the primary antibody, the PVDF membrane was washed three times with TBTS buffer on a horizontal shaker for 10 min each time, and then the secondary antibody (1:5000) was added and incubated at room temperature for 1 h.

[0055] 2.10 ECL Chemiluminescence Detection: After removing the secondary antibody, wash the PVDF membrane four times with TBTS buffer on a horizontal shaker for 8 minutes each time; place the PVDF membrane in a plastic film and mix ECL chemiluminescence solution A and B at a 1:1 (V / V) ratio; in a dark room, evenly coat an appropriate amount of the ECL chemiluminescence mixture onto the surface of the PVD membrane, expose it in the dark room, and adjust the exposure time according to the signal strength; finally, develop and fix the film sequentially.

[0056] The experimental results showed that the target flavonoid glycosides could inhibit iNOS expression in a concentration-dependent manner, suggesting that their anti-inflammatory mechanism may involve inhibiting the release of the inflammatory factor NO and regulating the pro-inflammatory signaling pathway of iNOS protein expression.

[0057] Based on the above, the advantages of this invention are that it uses dried flowers of *Trollius chinensis* (Ranunculaceae), and obtains a novel flavonoid C-glycoside compound through solvent extraction, macroporous adsorption resin column chromatography, silica gel column chromatography, polyamide column chromatography, ODS column chromatography, and preparative liquid chromatography. Pharmacological activity studies show that this compound inhibits NO production in LPS-induced RAW264.7 macrophages; Western blotting results show that this compound can downregulate the expression of iNOs, a related protein in the inflammatory signaling pathway. These results indicate that this compound has certain anti-inflammatory activity and is of great significance for the development of anti-inflammatory drugs.

[0058] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A flavonoid glycoside compound, characterized in that: The structural formula of the flavonoid glycosides is as follows: 。 2. The use of the flavonoid glycosides according to claim 1 in the preparation of anti-inflammatory drugs.

3. The method for preparing flavonoid glycosides according to claim 1, characterized in that, Includes the following steps: The process includes: Step 1, alcohol extraction of the raw material; Step 2, resin column chromatography; Step 3, silica gel column chromatography; Step 4, polyamide column chromatography; Step 5, ODS column chromatography; and Step 6, obtaining the target compound. In step one above, the dried golden lotus flower is extracted with an ethanol-water solution of 60% to 99% by volume, the soaking liquid is recovered and concentrated under reduced pressure to obtain crude ethanol extract. In step two above, the crude ethanol extract obtained in step one is dissolved in an appropriate amount of water, centrifuged and filtered, and the filtrate is subjected to ethanol-water gradient elution by macroporous resin column chromatography to obtain five fractions Fr.A to Fr.E, with ethanol to water volume ratios of 0:1, 3:7, 5:5, 7:3, and 95:5, respectively. In step three above, the fraction Fr.C from step two is subjected to silica gel column chromatography and eluted with a gradient of dichloromethane and methanol to obtain 11 fractions Fr.C to Fr.C11, with volume ratios of dichloromethane and methanol of 98:2, 93:7, 9:1, and 0:1, respectively. In step four above, the fraction Fr.C6 from step three is subjected to polyamide column chromatography and eluted with a methanol and water gradient to obtain eight fractions Fr.C6A to Fr.C6H, with methanol and water volume ratios of 1:4, 2:3, and 1:0, respectively. In step five above, the fraction Fr.C6E from step four is subjected to ODS column chromatography and eluted with a methanol and water gradient to obtain five fractions Fr.C6E1 to Fr.C6E5, with methanol and water volume ratios of 1:4, 1:3, 3:7, 1:1, and 1:0, respectively. In step six above, the target flavonoid glycosides are obtained by separating the fraction Fr.C6E4 from step five. The separation method is HPLC high performance liquid chromatography. The conditions of the HPLC high performance liquid chromatography are: ODS Rp-18, 5μm, 250×10mm, 35%MeOH / H2O, 5mL / min.

4. The preparation method according to claim 3, characterized in that: In step one, the dried golden lotus flowers are pre-chopped.

5. The preparation method according to claim 3, characterized in that: In step one, the alcohol extraction process specifically involves: heating and reflux extraction 2 to 4 times, each time for 1 to 3 hours.

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