A compound extracted and isolated from Codonopsis pilosula and its application in the preparation of anti-aging drugs.

Abstract

This invention relates to the field of pharmaceutical compound technology, specifically disclosing a compound extracted and isolated from Codonopsis pilosula and its application in the preparation of anti-aging drugs. The compound is named Codonopsis pilosula glycoside H. This invention employs modern spectroscopic techniques such as 1D-NMR, 2D-NMR, and high-resolution mass spectrometry to identify the structure of the isolated monomeric compound and deduce its molecular structure. Enzyme activity experiments show that the new compound has good PI3K inhibitory activity. Molecular docking results indicate that the new compound has good binding activity with the target protein, with hydrophobic interactions being predominant. Detection of SA-β-galactosidase staining in 2BS cells by the new compound demonstrates that the compound possesses good anti-aging activity and shows promising application prospects in anti-aging.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compound technology, specifically to a compound extracted and isolated from Codonopsis pilosula, namely codonopsis glycoside H (i.e. (8R,9R,2E,6Z,10E)-tetradeca-2,6,10-triene-4-yne-8,14-diol-9-β-D-glucopyranoside), and the application of this compound in the preparation of anti-aging drugs. Background Technology

[0002] Codonopsis tangshen Oliv. is one of the plants from which Codonopsis pilosula is derived. It is often used to treat symptoms such as spleen and lung qi deficiency, sallow complexion, and internal heat and thirst.

[0003] PI3K (phosphatidylinositol kinase) is a dimer composed of the regulatory subunit p85 and the catalytic subunit p110. When it binds to growth factor receptors (such as EGFR), it alters the protein structure of Akt and activates it, then activates or inhibits a series of downstream substrates through phosphorylation, such as apoptosis-related proteins Bad, Caspase 9, and mammalian target of rapamycin (mTOR), thereby regulating cell proliferation, differentiation, apoptosis, and migration phenotypes. Studies have shown that inhibiting PI3K activity can effectively delay aging and regulate lifespan. Therefore, drug screening targeting PI3K kinase may be of great significance in aging-related research.

[0004] In Chinese patent publications, patent application CN107056963A discloses Codonopsis pilosula pectin polysaccharide CPP1c and CPP1a, particularly the application of CPP1c in the preparation of drugs that inhibit cancer cell growth, promote cancer cell apoptosis, inhibit cancer cell metastasis, arrest cancer cell cycle, promote the expression of cancer cell apoptosis proteins, and inhibit the expression of cancer cell anti-apoptotic proteins, demonstrating significant medicinal and economic value. Patent CN109674810A discloses the use of Codonopsis pilosula polysaccharides in the preparation of drugs for treating anemia. The efficacy examples of this invention demonstrate that Codonopsis pilosula polysaccharides have positive therapeutic effects on blood deficiency and anemia, thus Codonopsis pilosula has excellent medical application prospects in the treatment of anemia. The aforementioned patent documents and other literature reporting on the related biological activities of Codonopsis pilosula mainly focus on its saccharide compounds, with few reports on the anti-aging effects of Codonopsis pilosula alkynol glycosides. Therefore, the extraction and isolation of alkynol glycosides from Codonopsis pilosula and the study of their pharmacological activities are of great significance. 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 new compound from Codonopsis pilosula and provide its anti-aging pharmaceutical uses.

[0006] To achieve the above-mentioned objectives of this invention, a new compound was extracted from Codonopsis pilosula:

[0007] Formula (1): Chemical formula is C 20 H 30 O8, chemically named (8R,9R,2E,6Z,10E)-tetradeca-2,6,10-triene-4-yne-8,14-diol-9-β-D-glucopyranoside, is named Codonopsis pilosula glycoside H, with the following structural formula:

[0008]

[0009] The method for extracting the above-mentioned new compound formula (1) from Codonopsis pilosula is as follows:

[0010] The dried root of Codonopsis pilosula was pulverized and extracted and concentrated by percolation with 95 v / v% ethanol and 60 v / v% ethanol, respectively. Then, it was extracted with petroleum ether, ethyl acetate and water-saturated n-butanol. The n-butanol extract was then purified by macroporous resin, normal phase silica gel column chromatography, Sephadex LH-20, reverse phase silica gel column chromatography and ODS-HPLC to obtain a new compound.

[0011] Furthermore, the novel compound of this invention exhibits good inhibitory activity against PI3K, with an IC50 value of 5.16 ± 0.35 μmol / L. The active novel compound extracted and isolated by this invention can be used to prepare anti-aging pharmaceuticals and health products, and is preferably used as an inhibitor of PI3K.

[0012] The beneficial effects of this invention are:

[0013] This invention extracts and isolates a novel compound from Codonopsis pilosula. This new compound belongs to the class of alkynyl glycosides and has good inhibitory activity against PI3K enzyme, thus it can be used to prepare anti-aging drugs and health products. Attached Figure Description

[0014] Figure 1 The above is the HR-ESI-MS spectrum of the new compound obtained in Example 1 of this invention;

[0015] Figure 2 The new compound obtained in Example 1 of this invention 1 H-NMR spectrum;

[0016] Figure 3 The new compound obtained in Example 1 of this invention 13 C-NMR spectrum;

[0017] Figure 4The DEPT 135° NMR spectrum of the new compound obtained in Example 1 of this invention;

[0018] Figure 5 The nuclear magnetic resonance HMQC spectrum of the new compound obtained in Example 1 of this invention;

[0019] Figure 6 The nuclear magnetic resonance HMBC spectrum of the new compound obtained in Example 1 of this invention;

[0020] Figure 7 The image shows the COSEY NMR spectrum of the new compound obtained in Example 1 of this invention.

[0021] Figure 8 The ECD spectrum of the new compound obtained in Example 1 of this invention is shown below.

[0022] Figure 9a and Figure 9b The figures shown are three-dimensional and two-dimensional images of the new compound obtained in Example 1 of this invention docking with PI3K.

[0023] Figure 10 The image shows a micrograph of the staining experiment measured in Example 4.

[0024] Figure 11 This is a bar chart of the SA-β-galactosidase staining experiment; where * indicates a difference compared to the control group. *** p < 0.001; # indicates that compared with the model group, ## p<0.01. Detailed Implementation

[0025] The applicant will now provide a clear and complete description of the technical solution of the present invention in conjunction with the embodiments and accompanying drawings.

[0026] In the following examples, Codonopsis pilosula was collected from Shennongjia, Hubei Province. Unless otherwise specified, all materials and reagents used were ordinary commercial reagents with analytical purity.

[0027] Example 1: Preparation of compound of formula (1)

[0028] Step 1: Take 16.0 kg of dried Codonopsis tangshen Oliv. root, crush it, and extract it successively with 95 v / v% ethanol and 60 v / v% ethanol by percolation. Combine the extracts and concentrate them to obtain 6 kg of total extract.

[0029] Step 2: After suspending the total extract obtained in Step 1 in water, extract it sequentially with petroleum ether, ethyl acetate, and water-saturated n-butanol to obtain petroleum ether extract (169.0 g), ethyl acetate extract (98.0 g), and n-butanol extract (691.0 g), respectively.

[0030] Step 3: Take 691.0 g of the n-butanol layer extract from Step 2, pass it through HP-20 macroporous adsorption resin, and perform gradient elution with water-ethanol. The water:ethanol volume ratios are 1:0, 7:3, 5:5, 3:7, 1:9, and 0:1, respectively. Collect the eluent with a water:ethanol volume ratio of 7:3, label it Fr.2, and concentrate it to dryness under reduced pressure for later use.

[0031] Step 4: Fraction Fr.2 obtained in Step 3 was separated by normal-phase silica gel (200-300 mesh) column chromatography using a gradient elution of dichloromethane:methanol:water (95:5:0-2:8:0.8), with volume ratios of dichloromethane:methanol:water being 95:5:0, 93:7:0, 9:1:0, 85:15:0, 8:2:0, 7:3:0.3, 6:4:0.4, 5:5:0.5, 4:6:0.6, 3:7:0.7, and 2:8:0.8. The eluent with a dichloromethane:methanol:water volume ratio of 8:2:0 was collected and labeled Fr.2.5, then concentrated to dryness under reduced pressure. Fraction Fr.2.5 was then subjected to Sephadex chromatography. LH-20 separation was performed using pure methanol as the elution system. The eluent volume was 2.0 column volumes. The eluent from 0.5 to 1.0 column volumes was collected and labeled Fr.2.5.2. The eluent was then concentrated to dryness under reduced pressure.

[0032] The fraction Fr.2.5.2 obtained in steps 5 and 4 was separated by reversed-phase silica gel (50 μm) column chromatography with a methanol:water gradient elution at volume ratios of 5:95, 3:7, 5:5, 7:3, 9:1, and 1:0. The eluent with a methanol:water volume ratio of 5:5 was collected and labeled Fr.2.5.2.3, and concentrated to dryness under reduced pressure. Fraction Fr.2.5.2.3 was purified by ODS-HPLC with AeCN-H2O as the eluent (AeCN-H2O volume ratio of 2:8), using a C-18 column (5 μm, 250 mm × 20 mm) at a flow rate of 3.0 mL / min. The eluent from 33 to 36.5 minutes was collected, concentrated and dried under reduced pressure to obtain a new compound (8.1 mg).

[0033] Structural identification: using modern spectroscopic techniques such as... 1 H NMR spectroscopy, 13The structure of the new compound obtained in step 5 was identified by 1200-2000 NMR spectroscopy, DEPT 135° NMR spectroscopy, two-dimensional NMR spectroscopy (HMQC, HMBC), high-resolution mass spectrometry (HR-ESI-MS), and ECD spectroscopy. The results are shown in the figure. Figure 1-8 ;

[0034] The new compound obtained in step 5 was identified as follows: HR-ESI-MS m / z 421.18304 [M+Na] + Its chemical formula is C 20 H 30 O8, named Codonopsis pilosula glycoside H, namely (8R,9R,2E,6Z,10E)-tetradeca-2,6,10-triene-4-yne-8,14-diol-9-β-D-glucopyranoside, has the following structural formula (1); its nuclear magnetic resonance spectrum data are shown in Table 1.

[0035] Table 1: 1 H-NMR and 13 C-NMR spectral data of 1( 1 H-NMR, 500MHz, DMSO; 13 C-NMR, 125MHz, DMSO, δppm, JHz)

[0036]

[0037]

[0038] The planar structure of the obtained compound is shown below:

[0039]

[0040] The HMBC spectra of the obtained compounds are shown below:

[0041]

[0042] The structure of the obtained compound is shown below:

[0043]

[0044] Example 2: To test the inhibitory activity of the new compound obtained in Example 1 against PI3K, the following enzyme activity experiment was performed:

[0045] Materials and reagents: The PI3Kα (p110α / p85) Assay Kit was purchased from BPS Bioscience, catalog number 79781. All experiments were performed in accordance with the kit's instruction manual.

[0046] Step 1: Thaw the 5x kinase assay buffer and ATP. Dilute the 5x kinase assay buffer with distilled water to prepare a 2.5x kinase assay buffer, and dilute the 500 μM ATP solution to 12.5 μM.

[0047] Step 2: Thaw PI3Kα on ice, calculate the amount of PI3Kα required for detection, and then dilute the enzyme to 0.5 ng / μl using the 2.5x kinase detection buffer prepared in Step 1.

[0048] Step 3: Add 5 μl of PI3K lipid substrate to each group. Add 5 μl of the prepared inhibitor (the new compound obtained in Example 1, quercetin) solution to the test group, and 5 μl of the prepared inhibition buffer without inhibitor to the control and blank groups. Add 5 μl of 12.5 μM ATP solution to each group. Add 10 μl of 2.5x kinase detection buffer to the blank group, and 10 μl of diluted PI3Kα enzyme to the control and test groups to start the reaction. Gently shake the wells and incubate at 30°C for 40 minutes.

[0049] Step 4: Thaw the ADP-Glo ​​reagent and add 25 μl of ADP-Glo ​​reagent to each well. Cover with aluminum foil and incubate at room temperature for 45 minutes. Thaw the kinase detection reagent and add 50 μl of kinase detection reagent to each well. Cover the plate with aluminum foil and incubate at room temperature for another 30 minutes. Record the fluorescence intensity of the microplate reader.

[0050] Table 2: Reagents added to the control group, test group, and blank group in the lipokinase reaction

[0051] Positive Control Test Inhibitor Blank PI3K lipid substrate 5μl 5μl 5μl Test Inhibitor - 5μl - Inhibitor buffer 5μl - 5μl Diluted ATP (12.5 μM) 5μl 5μl 5μl 2.5x Kinase buffer - - 10μl PI3Kα (0.5 ng / μl) 10μl 10μl - Total 25μl 25μl 25μl

[0052] Table 3: Inhibitory activity of the new compounds against PI3K enzyme

[0053] Compound numbering IC50 (μM) New compound 5.16±0.35 Quercetin 5.22±0.24

[0054] IC50 (μM) represents the concentration of the drug at which the enzyme activity inhibition rate is 50%, indicating its inhibitory activity on PI3K enzyme. Quercetin is the positive control drug for PI3K enzyme.

[0055] As shown in Table 3, the new compound exhibits good inhibitory activity against PI3K enzyme.

[0056] Example 3: To better understand the binding mode of the new compound obtained in Example 1 with PI3K, molecular docking was used for verification and explanation:

[0057] Step 1: Databases include UniProt (https: / / www.uniprot.org / ) and PDB (http: / / www.rcsb.org / ); software includes INdraw, SYBYL 2.0 (Tripos, USA), Discovery Studio 2017R2 Client (DS, developed by Accelrys, USA), and PyMOL.

[0058] 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 2.0 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.

[0059] Step 3: Download the crystal structure of the target protein PI3K from the PDB database (http: / / www.rcsb.org), modify the target protein using the Docking module in Application, add hydrogen and load AMBERFF99 charge, and 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.

[0060] Step 4: Using the Surflex-dock module of SYBYL 2.0 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.

[0061] Step 5: The molecular docking results were analyzed using the receptor-ligand interaction module in Discovery Studio software, and 3D and 2D renderings were created. The docking scores of the new compound with PI3K are shown in Table 4.

[0062] Table 4: Docking scores of new compounds with target proteins

[0063]

[0064] By studying the structural model of the new compound and PI3K ( Figure 9a and Figure 9b The compound forms hydrogen bonds with amino acid residues SER806, VAL882, ASP950, and ASP964, and hydrophobic interactions with amino acid residue TYR867. Therefore, the docking score of the novel compound with PI3K indicates that the novel compound of this invention has good binding activity to the target protein, and the novel compound may be a potential inhibitor of the PI3K enzyme.

[0065] Example 4: To further verify the anti-aging activity of the new compound obtained in Example 1, the effect of the new compound on SA-β-galactosidase staining of 2BS cells was tested.

[0066] The following are some of the reagents used: 2BS cells (Cell Bank of Wuhan University), Vitamin E (VitE), MTT (MCE), β-galactosidase staining kit (Shanghai Beyotime Biotechnology Co., Ltd.).

[0067] Step 1: Culture 2BS cells to the logarithmic growth phase using complete culture medium containing fetal bovine serum at 5% CO2 and 37°C.

[0068] Step 2: Take the well-grown 2BS cells from Step 1 and use 3 × 10⁻⁶ cells. 3 Cells were evenly seeded in 96-well plates. The cytotoxicity of different concentrations of the compound dissolved in DMSO was determined using the MTT assay to identify the non-toxic concentration. Specifically, 10 μl of 5 mg / ml MTT was added to each well of the culture plate, and the plates were incubated at 37°C for 4 h. The culture medium was then removed, and 150 μl of DMSO was added and shaken for 10 min. The absorbance of each well at 568 nm was measured using a microplate reader. The compound showed no toxicity to 2BS cells at a concentration of 20 μM.

[0069] Step 3: Seed the well-grown 2BS cells from Step 1 evenly into 96-well plates and culture for 24 hours. Establish a blank control group, a model group (0.2 mM H2O2), a drug group (0.2 mM H2O2 + new compound), and a positive control group (0.2 mM H2O2 + Vitamin E). Specifically: the model group, drug group, and positive control group were treated with 0.2 mM H2O2 for 2 hours, then replaced with fresh culture medium and cultured for another 5 days; the drug treatment groups (i.e., the drug group and the positive control group) received the corresponding concentration of drug 2 hours in advance. After group culture, aspirate the cell culture medium, wash once with PBS (0.01 mol / L, pH 7.4, the same below), add 500 μl of β-galactosidase staining fixative, and fix at room temperature for 15 minutes; aspirate the cell fixative, wash the cells three times with PBS; aspirate the PBS, add 500 μl of staining working solution to each well, and incubate overnight at 37°C; observe and photograph under a regular optical microscope.

[0070] The degree of cell senescence was detected using a β-galactosidase kit. Senescent cells were stained blue, and the more blue cells there were, the greater the degree of senescence. The experimental results showed that the number of stained cells after the intervention of the new compound was significantly less than that in the model group, indicating that the new compound can reduce the SA-β-galactosidase staining rate of H2O2-induced 2BS cells, suggesting that the new compound has good anti-aging activity.

Claims

1. A method for extracting the compound codonopsis glycoside H, characterized in that, The chemical formula of the compound Codonopsis pilosula glycoside H is C 20 H 30 O8, the structural formula is as follows: The extraction method includes the following steps: dried Codonopsis pilosula root is pulverized, extracted and concentrated by percolation with 95 v / v% ethanol and 60 v / v% ethanol, then extracted with petroleum ether, ethyl acetate and water-saturated n-butanol, and the obtained n-butanol layer extract is separated and purified by macroporous resin, normal phase silica gel column chromatography, Sephadex LH-20, reverse phase silica gel column chromatography and ODS-HPLC to obtain the compound; The specific conditions for separation and purification using the macroporous resin are as follows: HP-20 macroporous adsorption resin is used for gradient elution with water and ethanol, and the volume ratio of water to ethanol is 1:0, 7:3, 5:5, 3:7, 1:9, and 0:1, respectively. The specific steps for the separation and purification by normal-phase silica gel column chromatography are as follows: collect the eluent with a water:ethanol volume ratio of 7:3, concentrate it to dryness under reduced pressure, and separate it by 200-300 mesh normal-phase silica gel column chromatography with a dichloromethane:methanol:water gradient elution. The volume ratios of dichloromethane:methanol:water are 95:5:0, 93:7:0, 9:1:0, 85:15:0, 8:2:0, 7:3:0.3, 6:4:0.4, 5:5:0.5, 4:6:0.6, 3:7:0.7, and 2:8:0.

8. The specific steps for the Sephadex LH-20 separation and purification are as follows: collect the eluent with a volume ratio of dichloromethane:methanol:water of 8:2:0, concentrate it to dryness under reduced pressure, and separate it using Sephadex LH-20 with pure methanol as the elution system. The specific steps of the reversed-phase silica column chromatography separation and purification are as follows: collect the eluent of 0.5-1.0 column volume, concentrate it to dryness under reduced pressure, separate it by 50μm reversed-phase silica column chromatography, and elute with methanol:water gradient, with the volume ratio of methanol:water being 5:95, 3:7, 5:5, 7:3, 9:1, and 1:0 respectively. The specific steps of the ODS-HPLC separation and purification are as follows: collect the eluent with a methanol:water volume ratio of 5:5, concentrate it to dryness under reduced pressure, and purify it by ODS-HPLC. The eluent is AeCN-H2O with a volume ratio of 2:

8. The chromatographic column is a C-18 column with a size of 5μm, 250mm×20mm, and a flow rate of 3.0mL / min. Collect the eluent from the 33rd to 36.5th minute, concentrate and dry it under reduced pressure to obtain the compound Codonopsis pilosula glycoside H.

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