A compound extracted and separated from Codonopsis pilosula and application of the compound in preparing anti-inflammatory drugs

Abstract

The present invention relates to the technical field of medicinal compounds, and specifically discloses a compound extracted and separated from Codonopsis pilosula and the application of the new compound in the preparation of anti-inflammatory drugs, wherein the compound is named (7S, 8R)-7'(Z)-hierochin D. The present invention uses modern spectral techniques such as 1D-NMR, 2D-NMR, high-resolution mass spectrometry, etc. to structurally identify the isolated monomeric compound, and derive the molecular structure of the compound. The results of enzyme activity experiments show that the new compound has good inhibitory activity against AKT. The molecular docking results show that the new compound has good binding activity with the target protein AKT, and the interaction force is mainly hydrophobic.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and in particular to a compound (7S, 8R)-7'(Z)-hierochin D extracted and separated from Codonopsis pilosula and application of the compound in preparing anti-inflammatory drugs. Background Art

[0002] Sichuan Codonopsis pilosula is one of the commercial medicinal materials of Codonopsis pilosula, mainly produced in Hubei, Sichuan, Guizhou, Hunan and other places. It tastes sweet and is mild in nature. It belongs to the spleen and lung meridians. Its main effects are benefiting the lungs, strengthening the spleen, promoting body fluid, nourishing blood, etc. It is often used to treat spleen and lung qi deficiency, sallow complexion, internal heat and thirst and other diseases.

[0003] AKT is a serine / threonine kinase in the AGC kinase family, consisting of three subtypes, AKT1 / 2 / 3. It plays a key regulatory role in the PI3K / AKT pathway and plays an important role in various physiological activities such as cell apoptosis, proliferation and metabolism. When AKT is activated, it can bind to the IKK receptor to activate the NF-kB inflammatory pathway. Currently, only AKT1 is involved in the PI3K / AKT pathway. Studies have shown that when the PI3K / AKT / mTOR signaling pathway is activated, the content of proteins such as PI3K and AKT in the body increases significantly, releasing a large amount of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) to induce inflammatory diseases. Therefore, drug screening for AKT kinase targets may be of great significance for inflammatory diseases.

[0004] In the Chinese patent publications, the invention patent application with the publication number CN109350621A discloses the use of Codonopsis pilosula extract in the preparation of antioxidant active drugs for scavenging DPPH free radicals, indicating that 95% ethanol extract, water extract after alcohol extraction, crude polysaccharide, crude oligosaccharide, refined oligosaccharide, etc. of Codonopsis pilosula have antioxidant effects. Through the analysis of system component content and the comparison of component antioxidant activity, it can provide a basis for the discovery of pharmacological substances with antioxidant activity of Codonopsis pilosula. The invention with the publication number CN111195255A applied by Lanzhou University involves the use of Codonopsis pilosula oligosaccharides in the preparation of drugs for preventing or treating gastrointestinal damage caused by hypoxia. The invention proves that drugs containing Codonopsis pilosula oligosaccharides can improve spleen atrophy and lung enlargement caused by high-altitude hypoxia in rats, enhance their antioxidant level, improve inflammation, relieve intestinal mucosal damage, reduce EPO expression, and protein expression detection further shows that Codonopsis pilosula oligosaccharides can reduce the expression of HIF-1α in the duodenum of high-altitude hypoxic rats. The biological activities of Codonopsis pilosula reported in the above patent documents and other documents are mainly concentrated on the research of its extracts and the sugar compounds contained therein, while there are few reports on the anti-inflammatory effects of Codonopsis pilosula lignan compounds. Therefore, it is of great significance to extract and separate lignan compounds from Codonopsis pilosula and study their pharmacological activities. Summary of the invention

[0005] In view of the problems and shortcomings in the above-mentioned prior art, the purpose of the present invention is to extract a new compound from Codonopsis pilosula and provide its anti-inflammatory pharmaceutical use.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention extracts a new compound from Codonopsis pilosula:

[0007] Formula (1): Chemical formula is C 19 H 20 O6, molecular weight is 345.1333, chemical name is (7S,8R)-7'(Z)-hierochinD, structural formula is as follows:

[0008]

[0009] The method for extracting the new compound of formula (1) from Codonopsis pilosula comprises the following steps:

[0010] The dried root of Codonopsis pilosula was crushed, extracted and concentrated with 95 v / v% ethanol and 60 v / v% ethanol by percolation method, and then extracted with petroleum ether, ethyl acetate and water-saturated n-butanol in sequence. The petroleum ether layer and ethyl acetate layer extracts were combined, and then separated and purified by silica gel column chromatography, Sephadex LH-20 and ODS-HPLC in sequence to obtain a new compound of formula (1).

[0011] In addition, the new compound of the present invention has good inhibitory activity on AKT, and its IC50 is 3.53±0.66μmol / L. The active new compound extracted and separated by the present invention can be used to prepare anti-inflammatory drugs, preferably as an inhibitor of AKT.

[0012] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0013] The present invention extracts and separates a new compound from Codonopsis pilosula, and the new compound belongs to the lignan compound (benzotetrahydrofuran type), has good inhibitory activity on AKT enzyme, and can be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the HR-ESI-MS spectrum of the new compound of the present invention;

[0015] Figure 2 The new compound of the present invention 1 H-NMR spectrum;

[0016] Figure 3 The new compound of the present invention 13 C-NMR spectrum;

[0017] Figure 4 DEPT135°NMR spectrum of the new compound of the present invention;

[0018] Figure 5 is the nuclear magnetic resonance HMQC spectrum of the new compound of the present invention;

[0019] Figure 6 is the nuclear magnetic resonance HMBC spectrum of the new compound of the present invention;

[0020] Figure 7 is the nuclear magnetic resonance NOSEY spectrum of the new compound of the present invention;

[0021] Figure 8 It is the ECD spectrum of the new compound of the present invention;

[0022] Figure 9a and Figure 9b They are three-dimensional and two-dimensional effect diagrams of the new compound of the present invention and the target protein AKT, respectively. DETAILED DESCRIPTION

[0023] The applicant will now provide a clear and complete description of the technical solution of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings.

[0024] In the following examples, Codonopsis pilosula was collected from Shennongjia, Hubei Province, and the materials and reagents used, unless otherwise specified, were common commercial reagents with analytical purity.

[0025] Example 1: Preparation of the compound of formula (1)

[0026] Step 1, taking 16.0 kg of dried roots of Codonopsis tangshen Oliv., crushing them, extracting them with 95 v / v% ethanol and 60 v / v% ethanol by percolation method, combining the extracts, and concentrating them to obtain 6 kg of total extract;

[0027] Step 2, after suspending the total extract obtained in step 1 with water, extracting with petroleum ether, ethyl acetate, and water-saturated n-butanol in sequence to obtain a petroleum ether extract (169.0 g), an ethyl acetate extract (98.0 g), and an n-butanol extract (691.0 g), respectively;

[0028] Step 3, the combined ethyl acetate and petroleum ether extracts (267.0 g) obtained in step 2 were subjected to silica gel column chromatography, and gradient eluted with PE-EtOAc (20:1-1:1), with volume ratios of 20:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, and 2.1 L of the eluate with a volume ratio of petroleum ether: ethyl acetate of 1:1 was collected and numbered as Fr.7, and concentrated to dryness under reduced pressure for later use;

[0029] Step 4: The component Fr.7 obtained in step 3 was separated by silica gel column chromatography and eluted with a gradient of dichloromethane:methanol (500:1-9:1) in the following volume ratios: 500:1, 200:1, 99:1, 98:2, 97:3, 95:5, 9:1, and 0.6 L of the eluate with a dichloromethane-methanol volume ratio of 9:1 was collected and numbered as Fr.7.4, and concentrated to dryness under reduced pressure;

[0030] Step 5: The component Fr.7.4 obtained in step 4 was separated by Sephadex LH-20, using chloroform-methanol (volume ratio 1:1) as the elution system, and the amount of eluent was 1.5 column volumes. The eluent of 0.9-1.2 column volumes was collected and numbered as Fr.7.4.5, and concentrated to dryness under reduced pressure;

[0031] The component Fr.7.4.5 obtained in step 6 and step 5 was purified by ODS-HPLC with MeOH-H2O (MeOH:H2O volume ratio 30:70) as the eluent, C-18 column (5μm, 250mm×20mm), flow rate 3.0mL / min, and the eluate from 21 to 22.3 minutes was collected and concentrated under reduced pressure to obtain a new compound of formula (1) (2.5mg).

[0032] Structural identification: Using modern spectroscopic techniques such as 1 H NMR spectroscopy, 13 The new compound obtained in step 6 was structurally identified by C NMR, DEPT 135° NMR, two-dimensional NMR (HMQC, HMBC, NOSEY), high-resolution mass spectrometry (HR-ESI-MS), and ECD spectroscopy. Figure 1-8 .

[0033] After identification, the molecular weight of the new compound obtained in step 6 is 345.1333 and the chemical formula is C 19 H 20 O6, named (7S,8R)-7'(Z)-hierochin D, has the following structural formula (1); its nuclear magnetic resonance spectrum data are shown in Table 1. Table 1: Compound of formula (1) 1 H and 13 C NMR data ( 1 H-NMR, 500 MHz; 13 C-NMR,150MHz,in CD3OD,δppm,J Hz)

[0034]

[0035] The planar structure of the obtained compound is shown below:

[0036]

[0037] The HMBC spectrum of the obtained compound is shown below:

[0038]

[0039] The relative configuration determined by the NOESY spectrum of the obtained compound is as follows:

[0040]

[0041] The structure of the resulting compound is shown below:

[0042]

[0043] Example 2: To detect the inhibitory activity of the new compound obtained in Example 1 on Protein kinase B (AKT), the following enzyme activity experiment was performed:

[0044] Materials and reagents: AKT kinase activity kit was purchased from Enzo Life Sciences, with the catalog number ADI-EKS-400A. The following experiments were performed according to the product manual of the kit.

[0045] The experimental principle of the AKT enzyme activity experiment is as follows: add the test compound and kinase to a 96-well plate coated with a substrate synthetic peptide, add ATP to stimulate the reaction, and phosphorylate the substrate; after incubation for a period of time, stop the reaction, add the primary antibody (Phosphospecific Substrate Antibody) to the 96-well plate, which can specifically bind to the phosphorylated substrate; then add the secondary antibody (Anti-Rabbit IgG:HRP Conjugate), which binds to the phosphospecific antibody (primary antibody); use tetramethylbenzidine (TMB) for color development, and the color is positively correlated with the PKB enzyme activity. Use the stop solution to stop the color development reaction, and use a microplate reader to measure the absorbance value at 450nm.

[0046] Step 1. Add kinase dilution buffer (50 μL) to a 96-well plate containing PKB substrate, place at room temperature for 10 minutes, and then aspirate the liquid in each well. Then, according to the blank group, inhibitor group, and model group, 30 μL of kinase dilution buffer, 15 μL of activated PKB+15 μL of inhibitor, and 15 μL of activated PKB+15 μL of kinase dilution buffer were added to the corresponding wells, wherein the inhibitors were the compounds obtained in Example 1 and H-89. Except for the blank group, 10 μL of diluted ATP (1 mg / mL) was added to each well to start the reaction, and incubated at 30°C for 90 minutes. The contents of each well were emptied to terminate the reaction.

[0047] Step 2: Except for the blank group, 40 μL of phosphorylation-specific substrate antibody (primary antibody) was added to each well. After incubation at room temperature for 60 min, the liquid in each well was aspirated and the plate was washed 4 times with 100 μL of 1× washing buffer.

[0048] Step 3. Except for the blank group, add 40 μL of anti-rabbit IgG:HRP conjugate (secondary antibody, 1 μg / mL) diluted in antibody diluent to each well. After incubation at room temperature for 30 min, aspirate the liquid in each well and wash the plate 4 times with 100 μL 1× washing buffer.

[0049] Step 4: Add 60 μL of TMB substrate to each well and incubate at room temperature for 60 min. Then add stop solution 2 (20 μL) to each well to terminate the reaction. Measure the absorbance at 450 nm using an ELISA reader.

[0050] Table 2: Reagents added to the blank group, inhibitor group, and model group

[0051]

[0052] Table 3: Inhibitory activity of new compounds on AKT enzyme

[0053] Compound No. IC50(μM) Example 1 New compound obtained 3.53±0.66 H-89 3.97±0.97

[0054] IC50 (μM) is the concentration of the drug when the enzyme activity inhibition rate is 50%, which is used to indicate the inhibitory activity of the AKT enzyme. H-89 is the positive control drug for the AKT enzyme.

[0055] Example 3: In order to better understand the binding mode of the new compound obtained in Example 1 with AKT, a molecular docking method is now used for verification and interpretation:

[0056] Step 1. Databases include UniProt database (https: / / www.uniprot.org / ) and PDB database (http: / / www.rcsb.org / ); software include ChemOffice2010 (PerkinElmer, USA), SYBYL 1.0 software (Tripos, USA), Discovery Studio 2017R2 Client (DS, developed by Accelrys, USA), and PyMOL.

[0057] Step 2: Use ChemDraw software to draw the structural formula of the new compound and save it in mol2 format. Import the mol2 format structural formula of the new compound into SYBYL 1.0 software, use the molecular mechanics program Minimize to optimize the structure, assign the Tripos force field and load the Gasteiger-Huckel charge, save the optimized stable conformation in mol2 format, and establish a ligand small molecule compound library to prepare for molecular docking.

[0058] Step 3: Download the crystal structure of the target protein AKT from the PDB database (http: / / www.rcsb.org), modify, hydrogenate and load the AMBER FF99 charge on the target protein using the Docking module in the Application, determine the active site of the docking according to the ligand in the target protein complex, and save the processed protein to prepare for subsequent molecular docking studies.

[0059] Step 4: Use the Surflex-dock module of SYBYL 1.0 software to carry out molecular docking between the ligand small molecule compound library and the target protein. The docking result is given by the scoring function Total Score and saved in mol2 format. The ligand molecules are screened using the Total Score scoring function of the SYBYL molecular docking module. The Total Score scoring function comprehensively considers factors such as polarity, hydrophobicity, enthalpy and solvation. The larger the value, the more stable the docking complex, indicating that the matching and binding effect between the small molecule compound and the macromolecular protein is better.

[0060] Step 5: The receptor-ligand interactions module in the Discovery Studio software was used to analyze the molecular docking results and to produce three-dimensional and two-dimensional effect diagrams. The docking scores of the new compounds and AKT are shown in Table 4.

[0061] Table 4: Docking scores of new compounds and target proteins

[0062]

[0063] By studying the structural model of new compounds and AKT ( Figure 9a , 9b ). The compound forms hydrogen bonds with amino acid residues Glu228, Glu278, Thr291, and ASP292, and forms hydrophobic interactions with amino acid residues Val164, Ala177, and Met227. Therefore, combined with the docking scores of the new compound and AKT, it is shown that the new compound of the present invention has good binding activity with the target protein, and the new compound may be a potential inhibitor of AKT enzyme.

Claims

1. Use of the compound (7S,8R)-7'(Z)-hierochin D in the preparation of an AKT inhibitor, wherein the chemical formula of the compound is C 19 H 20 O6, the structural formula is as follows:

2. A method for extracting the compound (7S,8R)-7'(Z)-hierochin D, comprising the following steps: Take the dried root of Codonopsis pilosula, crush it, extract and concentrate it with 95v / v% ethanol and 60v / v% ethanol by percolation method, extract it with petroleum ether, ethyl acetate and water-saturated n-butanol in turn, and combine the obtained petroleum ether layer and ethyl acetate layer extracts; The combined ethyl acetate and petroleum ether extracts were subjected to silica gel column chromatography, with a petroleum ether-ethyl acetate system for gradient elution. The volume ratios of petroleum ether to ethyl acetate were 20:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, and 1:1, and the eluate with a volume ratio of petroleum ether to ethyl acetate of 1:1 was collected and numbered as Fr.7; The component Fr.7 was separated by silica gel column chromatography and eluted with a dichloromethane-methanol system gradient, the volume ratio of dichloromethane to methanol was: 500:1, 200:1, 99:1, 98:2, 97:3, 95:5, 9:1, and the eluate with a volume ratio of dichloromethane to methanol of 9:1 was collected and numbered as Fr.7.4; The component Fr.7.4 was separated by Sephadex LH-20, using chloroform-methanol with a volume ratio of 1:1 as the elution system, and the amount of eluent was 1.5 column volumes. The eluent from 0.9 to 1.2 column volumes was collected and numbered as Fr.7.4.5; Component Fr.7.4.5 was purified by ODS-HPLC. The eluent was methanol-water in a volume ratio of 30:

70. The chromatographic column was a C-18 column with a specification of 250 mm×20 mm×5 μm. The flow rate was 3.0 mL / min. The eluate from 21 to 22.3 minutes was collected, concentrated and dried to obtain the compound.

Images