Extracted and separated compound from nymphaea lotus and preparation method and application thereof

Abstract

The application relates to the technical field of medicinal compounds, and particularly discloses a compound 7alpha, 8alpha-epoxyblumenol C extracted and separated from Nymphaea lotus, and a preparation method and application of the compound. The application adopts modern spectrum technologies such as 1D-NMR, 2D-NMR, high-resolution mass spectrometry and the like to identify the structure of a monomer compound separated, and deduces the molecular structure of the compound. Enzyme activity experiment results show that the compound has good SIRT1 agonistic activity. Molecular docking results show that the compound has good binding activity with a target protein, and the interaction force is mainly hydrogen bond and hydrophobic force. The results show that the compound has good anti-inflammatory effect, and has good application prospect in treating inflammation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compound technology, specifically to a compound extracted and isolated from the pubescent-leaved water lily, its preparation method, and its application. Background Technology

[0002] The pubescent toothed water lily is a plant belonging to the genus Nymphaeum in the family Nymphaeaceae. Nymphaea lotus var. pubescens (Dried flowers)

[0003] Sirtuins deacetylase uses nicotinamide adenine dinucleotide (NAD) + As a cofactor, it catalyzes the deacetylation of proteins, while simultaneously releasing NAD+. + It decomposes into nicotinamide (NAM) and 2'-O-acyl-ADP-ribose (OAADPr). Seven Sirtuins isoforms (SIRT1~SIRT7) have been identified in the human body, each with different subcellular localization and functional characteristics. Among them, Silent Information Regulator 1 (SIRT1) is widely distributed in various tissues such as the brain, heart, kidneys, liver, retina, uterus, skeletal muscle, blood vessels, and adipose tissue. It is mainly located in the cell nucleus and can enter the cytoplasm via nucleoplasmic transport, playing a key regulatory role in various physiological processes in the human body. Studies have found that SIRT1 is a nicotinamide adenine dinucleotide (NAD) dependent... +SIRT1, a class III histone deacetylase, can regulate the hyperacetylation of various factors, including HMGB1, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and NF-κB. SIRT1, in particular, can regulate the inflammatory response by inhibiting the extracellular release of HMGB1. PGC-1α also participates in the regulation of the inflammatory response; it can induce skeletal muscle cells to produce secretory phosphoprotein 1, thereby recruiting macrophages to secrete monocyte chemoattractant protein 1, and simultaneously aggregate endothelial cells and smooth muscle cells to induce angiogenesis, ultimately contributing to the formation of an anti-inflammatory microenvironment. Furthermore, NF-κB activity is tightly regulated by SIRT1. SIRT1 can directly deacetylate the lysine residue at position 310 of the p65 subunit of NF-κB, preventing the RelA / p65 subunit from binding to IκBα. This prevents NF-κB from entering the nucleus and inducing the expression of related genes, thereby inhibiting NF-κB activity and the transcription of downstream genes, ultimately reducing the production of inflammatory factors such as TNF-α and IL-1β. Based on the crucial role of SIRT1 in inflammation regulation, the development of potent and highly selective SIRT1 agonists, which promote SIRT1 deacetylase activity, can effectively regulate inflammatory responses and thus be applied to the treatment of related inflammatory diseases.

[0004] In Chinese patent publications, CN116554263A discloses a class of SIRT1 agonists. These compounds exhibit excellent subtype selectivity for SIRT1 and can be used for interventional treatment of conditions such as inflammation, COVID-19 infection, sepsis, diabetes, tumors, cardiovascular diseases, acute kidney injury, nerve injury, and neurodegenerative diseases. CN118027125A reports triterpenoid compounds that can activate SIRT1, mediating heat shock factor-1 deacetylation, upregulating heat shock protein expression, and inhibiting cardiomyocyte apoptosis. These compounds could serve as lead compounds for the treatment of myocardial ischemia-reperfusion injury and show promising research potential. Existing patents and related studies have confirmed the significant potential of targeted regulation of SIRT1 in anti-inflammatory drug development. However, current research on the chemical components and anti-inflammatory activities of Nymphaeus pubescens extract remains relatively scarce. Therefore, the extraction, separation, and anti-inflammatory pharmacological activity evaluation of active ingredients from Nymphaeus pubescens are of significant research value and practical importance for discovering novel natural anti-inflammatory lead compounds. Summary of the Invention

[0005] In view of the problems and deficiencies in the prior art, the purpose of this invention is to extract a compound from Nymphoides pubescens, and to provide its preparation method and anti-inflammatory pharmaceutical use.

[0006] To achieve the above-mentioned objectives of this invention, the present invention provides compounds extracted from *Nymphaea pubescens*:

[0007] The chemical formula is C 13 H 20 O3, the structural formula is as follows:

[0008] (1)

[0010] The method for preparing the compound shown in formula (1) from the pubescent toothed water lily is as follows:

[0011] Step 1: Take the dried flowers of the soft-haired toothed water lily, crush them, and extract them sequentially with 90% v / v and 60% v / v ethanol using the percolation method. Combine the extracts and concentrate them to obtain the total extract.

[0012] Step 2: Add water to the total extract obtained in Step 1 to make a suspension, extract with petroleum ether, discard the petroleum ether phase and retain the aqueous phase; continue to extract the aqueous phase with ethyl acetate, collect the ethyl acetate phase, and concentrate under reduced pressure to obtain the ethyl acetate fraction.

[0013] Step 3: Load the ethyl acetate fraction obtained in Step 2 onto a silica gel column for chromatography. Elute with a gradient of petroleum ether-ethyl acetate system at volume ratios of 20:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 1:1. Collect the eluent with a petroleum ether-ethyl acetate volume ratio of 1:1 and concentrate to dryness under reduced pressure.

[0014] Step 4: Separate the solid obtained in Step 3 again by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol with volume ratios of 100:0, 99:1, 98:2, 97:3, and 95:5. Collect the eluent with a volume ratio of 98:2 and concentrate it to dryness under reduced pressure.

[0015] Step 5: Separate the solid obtained in Step 4 using a Sephadex LH-20 gel column, elute isocratically with methanol, collect 0.1~0.5 column volume of eluent, and concentrate to dryness under reduced pressure.

[0016] Step 6: The solid obtained in Step 5 was purified by ODS reversed-phase high-performance liquid chromatography. The mobile phase was methanol-water, volume ratio 37:63, the column was a C18 column with a size of 5 μm, 250 mm × 10 mm, and the flow rate was 3.0 mL / min. The eluent corresponding to the retention time of 47-49 minutes was collected, concentrated and dried under reduced pressure to obtain the compound.

[0017] The application of this compound in the preparation of anti-inflammatory drugs.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0019] This invention extracts and isolates a novel compound from *Nymphaea pubescens*, which exhibits good SIRT1 agonist activity and EC50. 1.5 The value was 12.73±0.84 μM; this compound can induce NF-κB deacetylation, inhibit NLRP3 inflammasome activation, and weaken immune and inflammatory responses, and can be used to prepare anti-inflammatory drugs. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The HR-ESI-MS spectrum of the compound obtained in Example 1 of this invention;

[0022] Figure 2 The IR spectrum of the compound obtained in Example 1 of this invention;

[0023] Figure 3 The compound obtained in Example 1 of this invention 1 H-NMR spectrum;

[0024] Figure 4 The compound obtained in Example 1 of this invention 13 C-NMR spectrum;

[0025] Figure 5 The DEPT 135° NMR spectrum of the compound obtained in Example 1 of this invention;

[0026] Figure 6 The DEPT 90° NMR spectrum of the compound obtained in Example 1 of this invention;

[0027] Figure 7 The nuclear magnetic resonance HMQC spectrum of the compound obtained in Example 1 of this invention;

[0028] Figure 8 The nuclear magnetic resonance HMBC spectrum of the compound obtained in Example 1 of this invention;

[0029] Figure 9 The COSY NMR spectrum of the compound obtained in Example 1 of this invention;

[0030] Figure 10The NMR NOESY spectrum of the compound obtained in Example 1 of this invention;

[0031] Figure 11 This is a two-dimensional effect diagram of the compound obtained in Example 1 of the present invention and the target protein SIRT1;

[0032] Figure 12 This is a three-dimensional effect diagram of the compound obtained in Example 1 of the present invention and the target protein SIRT1. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] The reagents used in the following examples are all common commercial reagents with analytical purity.

[0035] Example 1: Preparation of compound of formula (1)

[0036] Step 1: Take 5.0 kg of dried flowers of the soft-haired toothed water lily, crush them through a 50-mesh sieve, and extract them sequentially with 90 v / v% ethanol (50 L) and 60 v / v% ethanol (51 L) by percolation. Combine the extracts and concentrate them to obtain a total extract of 1418 g.

[0037] Step 2: After suspending the total extract obtained in Step 1 in water, extract twice with petroleum ether, each time using 1.5L. After extraction, the aqueous phase is extracted five times with ethyl acetate, each time using 2.0L. Combine all the ethyl acetate extracts and concentrate under reduced pressure to obtain 244g of ethyl acetate fraction.

[0038] Step 3: Separate the ethyl acetate fraction (174g) obtained in Step 2 by silica gel (200-300 mesh) column chromatography. Elute sequentially with petroleum ether:ethyl acetate:methanol at volume ratios of 20:1:0, 10:1:0, 8:1:0, 6:1:0, 4:1:0, 3:1:0, 2:1:0, 1:1:0, 1:2:0, 1:3:0, 0:1:0, 0:95:5, 0:9:1, 0:8:2, 0:7:3, 0:6:4, and 0:5:5. Combine the results with thin-layer chromatography (TLC) detection. Combine the same components and collect the eluent with a petroleum ether:ethyl acetate:methanol volume ratio of 1:1:0. Concentrate to dryness under reduced pressure and label it Fr.3 for later use.

[0039] Step 4: Separate the fraction Fr.3 obtained in Step 3 by silica gel (200-300 mesh) column chromatography, using a gradient elution of dichloromethane:methanol (100:0-95:5) with volume ratios of 100:0, 99:1, 98:2, 97:3, and 95:5. Combine the results with thin-layer chromatography (TLC) detection, combine the same fractions, collect the eluent with a dichloromethane-methanol volume ratio of 98:2, concentrate it to dryness under reduced pressure, and label it Fr.3.2.

[0040] Step 5: Separate the fraction Fr.3.2 obtained in Step 4 using Sephadex LH-20, and perform isocratic elution with methanol as the eluent. The eluent volume is 1.5 column volumes. Collect the eluent from 0.1 to 0.5 column volumes, concentrate it to dryness under reduced pressure, and label it Fr.3.2.1.

[0041] Step 6: The fraction Fr.3.2.1 obtained in step 5 was purified by ODS-HPLC. The eluent was methanol-water (MeOH:H2O volume ratio of 37:63). The chromatographic column was a C-18 column (5μm, 250mm×10mm). The flow rate was 3.0mL / min. The eluent was collected and retained for 47 to 49 minutes. The eluent was concentrated and dried under reduced pressure to obtain a new compound (2.7mg), namely compound (1).

[0042] Structural identification: using modern spectroscopic techniques such as... 1 H NMR spectrum, 13 The structure of the new compound obtained in step 6 was identified using C10 NMR spectroscopy, DEPT 135°NMR spectroscopy, DEPT 90°NMR spectroscopy, two-dimensional NMR spectroscopy (HMQC, HMBC, COSY, NOESY), high-resolution mass spectrometry (HR-ESI-MS), and infrared spectroscopy (IR). The results are shown in the figure. Figure 1-10 ;

[0043] The new compound obtained in step 6 was identified as having an HR-ESI-MS m / z value of 225.1485 [M+H]. + Its chemical formula is C 13 H 20 O3, named 7α,8α-epoxyblumenol C, has the following structural formula (1); its nuclear magnetic resonance spectrum data are shown in Table 1.

[0044] Table 1: Compounds of Formula (1) 1 H NMR and 13 C NMR data ( 1 H NMR, 500MHz 13 C NMR, 125MHz, in CDCl3, δppm, J Hz)

[0045]

[0046] The structural formula of the new compound is shown in formula (1) below:

[0047] (1)

[0049] The HMBC spectrum of the new compound is shown in equation (2) below:

[0050] (2)

[0052] The NOESY spectrum of the new compound is shown in equation (3) below:

[0053] (3)

[0055] Example 2: To detect the agonistic activity of the new compound prepared in Example 1 against histone deacetylase (SIRT1), the following enzyme activity experiment was performed:

[0056] Materials and reagents: The SIRT1 activity assay kit used in the experiment was purchased from Abcam, catalog number AB156065. All experiments were conducted in accordance with the product manual of the kit.

[0057] Experimental setup: test sample group, solvent control group, positive control group, enzyme-free control group and development control group. The reagents added and their amounts are shown in Table 2.

[0058] Step 1: Thaw SIRT1 Assay Buffer, Fluoro Substrate Peptide, Fluoro Deacetylated, NAD, Developer, and Recombinant SIRT1 reagent at low temperature.

[0059] Step 2: Add ddH2O, SIRT1 Assay Buffer, Fluoro Substrate Peptide, Fluoro Deacetylated, and NAD to the wells of the microplates in different groups according to Table 2.

[0060] Step 3: Add DMSO solvent, the new compound dissolved in DMSO, and resveratrol positive control to the wells of the microplate respectively (the new compound and resveratrol positive control are set at six concentration gradients of 0.5, 1.5, 4.5, 13.5, 40.5, and 121.5 μmol / L respectively), and mix well.

[0061] Step 4: Add the Developer to each well of the microplate and mix thoroughly.

[0062] Step 5: Add 5 µL of Recombinant SIRT1 to the wells of the microplates in different groups according to Table 2 and mix thoroughly at room temperature (25°C) to start the reaction.

[0063] Step 6: Using a microplate fluorometer, under conditions of excitation wavelength of 350 nm and emission wavelength of 450 nm, read the fluorescence intensity every minute for 30 to 60 minutes. While maintaining a constant reaction rate (observe the time-fluorescence curve; if the curve shows no significant bend, the rate is considered constant), measure and calculate the reaction rate (fluorescence signal change rate, ΔRFU / min). Calculate the relative activity based on the reaction rate, and fit the dose-response curve to obtain the EC50 of the new compound and resveratrol. 1.5 value.

[0064] Table 2: Reagents added to the test sample group, solvent control group, positive control group, enzyme-free control group, and development control group

[0065]

[0066] Table 3: Agonistaltic activity of the new compounds against SIRT1 enzyme

[0067]

[0068] Among them, EC 1.5 (μM) is the concentration of the compound required to increase enzyme activity by 50% (i.e., relative activity of 150%) compared to the solvent control group, used to represent the agonistic activity against SIRT1 enzyme; resveratrol is the positive control drug for SIRT1 enzyme, as shown in Table 3, the new compound has good agonistic activity against SIRT1 enzyme.

[0069] Example 3: To better understand the binding mode of the new compound obtained in Example 1 with SIRT1, molecular docking methods are used for verification and explanation:

[0070] Step 1: Databases: UniProt database (https: / / www.uniprot.org / ), PDB database (http: / / www.rcsb.org / ); Software: InDraw (Shanghai Yinggu Information Technology Co., Ltd., China), SYBYL 1.0 software (Tripos, USA), Discovery Studio 2017R2 Client (DS, developed by Accelrys, USA), PyMOL.

[0071] Step 2: Use InDraw software to draw the structural formula of the new compound and save it in mol2 format. Import the mol2 format structure of the new compound into SYBYL 1.0 software, use the molecular mechanics program Minimize for structural optimization, apply a Tripos force field and load Gasteiger-Huckel charge, save the optimized stable conformation in mol2 format, and build a ligand small molecule compound library to prepare for molecular docking.

[0072] Step 3: Download the crystal structure of the target histone deacetylase (SIRT1) (PDB ID: 5BTR) from the PDB database. Modify, hydrogenate, and load AMBER-FF99 charge on the target protein using the Docking module in Application. Determine the docking active site based on the ligands in the target protein complex. Save the processed protein to prepare for subsequent molecular docking studies.

[0073] Step 4: Using the Surflex-dock module of SYBYL 1.0 software, perform molecular docking between the ligand small molecule compound library and the target protein. The docking results are given by the Total Score function and saved in mol2 format. The Total Score function of the SYBYL molecular docking module is used to screen ligand molecules. The Total Score function comprehensively considers factors such as polar interactions, hydrophobic interactions, enthalpy, and solvation. The higher the value, the more stable the docking complex, indicating a better matching and binding effect between the small molecule compound and the large protein molecule.

[0074] Step 5: Analyze the molecular docking results using the receptor-ligand interaction module in Discovery Studio and PyMOL software, and create two-dimensional and three-dimensional renderings. The docking scores of the new compound and the target protein are shown in Table 4.

[0075] Table 4: Docking scores of new compounds with target proteins

[0076]

[0077] The structural model of the new compound and SIRT1 was studied. Figure 11 and Figure 12The compound is stabilized through hydrogen bonding with THR209 and LYS444, and hydrophobic interactions with residues LEU202, PRO211, PRO212, LEU215, and ILE223. These hydrogen bonds and hydrophobic interactions play a crucial role in the binding of the novel compound to SIRT1. The docking score of the novel compound with SIRT1 indicates that the novel compound of this invention exhibits good binding activity to the target protein, and the novel compound may be a potential agonist of the SIRT1 enzyme.

[0078] Example 4: To further verify the anti-inflammatory activity of the new compound obtained in Example 1, the effect of the new compound on the NO release of RAW264.7 cells was tested.

[0079] The following are some of the reagents and instruments used: RAW264.7 cell line (China Center for Type Culture Collection); DMEM medium (Gibco); lipopolysaccharide (Sigma); fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.); NO detection kit (Shanghai Beyotime Biotechnology Co., Ltd.); sterile CO2 incubator (Heal Force Biomedical Technology Holdings Co., Ltd.); full-wavelength microplate reader (Thermo Fisher Scientific).

[0080] Step 1: RAW264.7 cells were cultured to the logarithmic growth phase in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. New compound and positive control solutions were prepared using DMSO, and the following experiments were then performed:

[0081] Step 2: The cytotoxicity of the compound was determined using the MTT assay. The compound at a final concentration of 50 μM showed no cytotoxic effect on RAW264.7 cells. Specifically:

[0082] The RAW264.7 cell suspension from step 1, grown in the logarithmic phase, was evenly seeded into 96-well plates (1×10⁻⁶ cells per well). 4 Cells per well (5 replicates per well) were cultured for 24 hours at 37°C with 5% CO2 before drug administration. A blank group (no LPS, no drug), a control group (final concentration 1 μg / mL LPS culture medium), and a drug administration group (final concentration 1 μg / mL LPS culture medium + final concentration 50 μM new compound solution) were set up and incubated at 37°C with 5% CO2 for 48 hours. The culture plate was then removed, and 20 μL of MTT solution (5 mg / mL) was added to each well. Incubation was continued at 37°C for another 4 hours before termination. The culture supernatant was carefully aspirated from the wells, and 150 μL of DMSO was added to each well. After shaking for 10 min, the absorbance (OD) of each well was measured at 490 nm using an automated microplate reader. Cell viability (%) = absorbance of experimental group (OD). 实验 ) / Control group absorbance (OD)对照 (×100%), survival rate ≥80%. Generally, compounds are considered to have no obvious toxic effects. The results show that the compound at a final concentration of 50 μM has no toxic effect on RAW264.7 cells.

[0083] Step 3: Detect NO release using the Griess method.

[0084] RAW264.7 cells in the logarithmic growth phase were seeded in 96-well plates (1×10⁻⁶ cells per well). 4 Cells / well were divided into a blank group (no LPS, no drug), a model group (1 μg / mL LPS), a solvent control group (1 μg / mL LPS + DMSO), a drug treatment group (1 μg / mL LPS + new compound), and a positive control group (1 μg / mL LPS + dexamethasone), with 5 replicates per group. Except for the blank group, the positive control group and the drug treatment group were incubated with the drug for 2 h, and then simultaneously stimulated with LPS at a final concentration of 1 μg / mL for 22 h, just like the model group. 50 μL of supernatant was taken, and 50 μL of Griess solution A and solution B were added according to the Griess method. The absorbance value was measured on an automated microplate reader (measured at 540 nm). Sodium nitrite standard was used simultaneously for the above experiment. A standard curve was plotted with the standard concentration as the x-axis and the OD value as the y-axis, and a linear regression equation was obtained. The nitrite content in the cell supernatant of each group was calculated according to the linear regression equation to characterize the nitric oxide level. The NO release inhibition rate was calculated as follows: NO release inhibition rate (%) = (C 模型组 -C 给药组 ) / (C 模型组 -C 空白组 ) × 100%, where C 模型组 C 给药组 C 空白组 The levels of nitrite (NO) in the cell supernatant of the model group, drug-treated group, and blank group were respectively... 2- Concentration; plot the compound concentration logarithm on the x-axis and NO release inhibition rate (%) on the y-axis, and fit the data to obtain IC50. 50 value.

[0085] Table 5: In vitro anti-inflammatory activity of the new compounds

[0086]

[0087] The results showed that the new compound could significantly downregulate NO release from inflammatory cells, and the IC50 value was significantly higher than that of other compounds. 50 The value was lower than that of the positive control drug dexamethasone, indicating that the new compound has good anti-inflammatory activity.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound as shown in formula (1), with the chemical formula C 13 H 20 O3, structural formula: （1）。 2. A method for preparing the compound according to claim 1, characterized in that, Includes the following steps: Step 1: Take the dried flowers of the soft-haired toothed water lily, crush them, and extract them sequentially with 90% v / v and 60% v / v ethanol by percolation. Combine the extracts and concentrate them to obtain the total extract. Step 2: Add water to the total extract obtained in Step 1 to make a suspension, extract with petroleum ether, discard the petroleum ether phase and retain the aqueous phase; continue to extract the aqueous phase with ethyl acetate, collect the ethyl acetate phase, and concentrate under reduced pressure to obtain the ethyl acetate fraction; Step 3: Load the ethyl acetate fraction obtained in Step 2 onto a silica gel column for chromatography, and elute with a gradient of petroleum ether-ethyl acetate system at volume ratios of 20:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 1:

1. Collect the eluent with a petroleum ether-ethyl acetate volume ratio of 1:1 and concentrate it to dryness under reduced pressure. Step 4: Separate the solid obtained in Step 3 again by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol, with the volume ratios of dichloromethane-methanol being 100:0, 99:1, 98:2, 97:3, and 95:5 respectively. Collect the eluent with a dichloromethane-methanol volume ratio of 98:2 and concentrate it to dryness under reduced pressure. Step 5: Separate the solid obtained in Step 4 using a Sephadex LH-20 gel column, elute isocratically with methanol, collect 0.1~0.5 column volume of eluent, and concentrate to dryness under reduced pressure; Step 6: The solid obtained in Step 5 was purified by ODS reversed-phase high-performance liquid chromatography. The mobile phase was methanol-water, volume ratio 37:63, the column was a C18 column with a size of 5 μm, 250 mm × 10 mm, and the flow rate was 3.0 mL / min. The eluent corresponding to the retention time of 47-49 minutes was collected, concentrated and dried under reduced pressure to obtain the compound.

3. The use of the compound of claim 1 in the preparation of anti-inflammatory drugs.

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