A sesquiterpenoid compound from Apricot-scented rabbit ear wind and its preparation method and application

Abstract

The present application belongs to the field of medical technology, and discloses a sesquiterpenoid compound in Apricot-scented Rabbit Ear Wind and its preparation method and application. The compound is named Ainslinine B. Apricot-scented Rabbit Ear Wind is extracted with alcohol, and petroleum ether, dichloromethane, ethyl acetate, and water are added for extraction. The water extraction part is first separated by resin, and then separated by silica gel column chromatography and preparative high performance liquid chromatography in sequence to obtain the target compound. The preparation method of the compound of the present invention is simple to operate, and the sesquiterpenoid compound can be separated and obtained. Experiments show that the compound of the present invention can effectively inhibit the proliferation of A549 cells (human non-small cell lung cancer cells), has a significant anti-tumor effect, and can be further used in the preparation of anti-lung cancer drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a sesquiterpenoid compound in apricot-scented radix serrata and a preparation method and application thereof. Background Art

[0002] At present, strategies for treating lung cancer include surgery, chemotherapy, radiotherapy, immunotherapy and targeted therapy, but these methods are often accompanied by significant side effects and may affect the patient's long-term quality of life. Therefore, the medical community has been looking for more effective treatments with fewer side effects. In recent years, researchers at home and abroad have found that active ingredients in traditional Chinese medicine have shown great potential in anti-lung cancer treatment. For example, epimedium polysaccharides, tanshinone I, astragaloside IV, etc., these ingredients can target the signaling pathways of lung cancer and improve the immune microenvironment of lung cancer by regulating immune cells in the tumor microenvironment, such as macrophages, natural killer cells, myeloid suppressor cells, T cells, regulatory T cells and dendritic cells. These active ingredients can enhance the function and activity of immune cells, enhance the body's immune function, and reverse the immunosuppressive state, thereby playing an important role in anti-lung cancer treatment. Therefore, discovering and developing new anti-lung cancer drugs from traditional Chinese medicine has become a hot topic in drug research today.

[0003] Apricot scented rabbit ear wind Ainsliaea fragrans Champ. is a perennial herbaceous plant of the genus Rhizoma in the Asteraceae family. The plant has the effects of clearing away heat and detoxifying, eliminating accumulation and dispersing knots, relieving cough and stopping bleeding. Summary of the invention

[0004] The purpose of the present invention is to conduct in-depth research on the chemical components of apricot-scented rabbit ear wind, provide a sesquiterpenoid compound in apricot-scented rabbit ear wind and a preparation method and application thereof, and the research finds that the sesquiterpenoid compound of the present invention has an anti-lung cancer effect.

[0005] The technical solution of the present invention is as follows:

[0006] A sesquiterpenoid compound in Apricot-scented rabbit ear wind is named Ainslinine B, and its structural formula is shown below:

[0007] .

[0008] The present invention also provides a method for preparing the sesquiterpenoid compound, comprising the following steps:

[0009] (1) Take the whole herb of Psoralea corylifolia and add ethanol solution for extraction to obtain an extract;

[0010] (2) Concentrate the extract until it has no alcohol taste, and then add petroleum ether, dichloromethane, ethyl acetate, and water to extract the extracts to obtain the extracts;

[0011] (3) The water extraction fraction obtained in step (2) was dissolved in methanol, separated by DAION HP20 macroporous adsorption resin, and eluted with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 90% ethanol aqueous solution in sequence to obtain corresponding fractions;

[0012] (4) taking the fraction eluted with 30% ethanol aqueous solution in step (3), dissolving it with methanol, dry-mixing it with silica gel (100-200 mesh), loading it onto a pre-filled silica gel (100-200 mesh) column, performing column chromatography separation, gradient eluting with dichloromethane-methanol (volume ratio of 100:0, 85:1, 50:1, 25:1, 10:1, 5:1, 0:100), and detecting it with high performance liquid chromatography, combining the fractions with similar liquid phase spectra to obtain 15 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0013] (5) The fraction 12 obtained in step (4) was dissolved in methanol and separated by preparative HPLC using acetonitrile-0.1% formic acid aqueous solution (volume ratio 20:80) as the mobile phase. The isocratic elution was performed at a flow rate of 10 ml / min with a retention time of 35 min to obtain the target compound.

[0014] Preferably, the extraction method in step (1) is cold soaking, percolation, microwave extraction, ultrasonic extraction or reflux extraction. More preferably, reflux extraction is performed twice, each time for 2 hours, and the extraction temperature is 70-90°C.

[0015] Preferably, the ethanol solution used in step (1) is an ethanol aqueous solution with a volume concentration of 5% to 95%, more preferably a 65% ethanol aqueous solution. The amount of ethanol aqueous solution added during extraction is 3 to 50 times the volume of the medicinal material, more preferably 10 times.

[0016] Preferably, in step (4), the mass ratio of the fraction obtained from the 30% ethanol aqueous solution elution portion to the silica gel used for sample mixing is 1:2.2.

[0017] The invention also provides application of the sesquiterpenoid compound in preparing anti-lung cancer drugs.

[0018] The invention also provides a pharmaceutical composition comprising the sesquiterpenoid compound.

[0019] The present invention also provides a pharmaceutical preparation, comprising a therapeutically effective amount of a sesquiterpenoid compound and a pharmaceutically acceptable carrier or excipient thereof.

[0020] Those skilled in the art can directly or indirectly add the compound to various commonly used pharmaceutically acceptable excipients required for preparing different dosage forms, such as fillers, disintegrants, lubricants, adhesives, etc., and prepare commonly used oral preparations or injection preparations using conventional pharmaceutical preparation methods.

[0021] Preferably, the oral preparation is a tablet, a capsule, a granule, a fat emulsion, a microcapsule, or a pill.

[0022] Preferably, the injection preparation is an injection solution or a powder injection.

[0023] The beneficial effects of the present invention are:

[0024] The preparation method of the compound of the invention is simple to operate and can separate and obtain a sesquiterpenoid compound in the apricot-scented sedge.

[0025] Experiments show that the compound of the present invention can effectively inhibit the proliferation of A549 cells (non-small cell lung cancer cells), has a significant anti-tumor effect, and can be further used in the preparation of anti-lung cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the chemical structural formula of the compound of the present invention;

[0027] Figure 2 is the mass spectrum of the compound of the present invention;

[0028] Figure 3 The compounds of the present invention 1 H NMR spectrum;

[0029] Figure 4 The compounds of the present invention 13 C NMR spectrum;

[0030] Figure 5 is the DEPT spectrum of the compound of the present invention;

[0031] Figure 6 is the HSQC spectrum of the compound of the present invention;

[0032] Figure 7 is the HMBC spectrum of the compound of the present invention;

[0033] Figure 8 The compounds of the present invention 1 H- 1 H COSY spectrum;

[0034] Figure 9 is the NOESY spectrum of the compound of the present invention;

[0035] Figure 10The figure shows the effect of the compounds of the present invention on the clone formation of A549 cells. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0038] Example 1 Preparation of Compounds

[0039] This embodiment provides a method for preparing a sesquiterpenoid compound in Psoralea corylifolia, the steps being:

[0040] (1) Take the whole herb of Psoralea corylifolia, add 10 times the volume of 65% ethanol solution, reflux and extract twice, each time for 2 hours, to obtain the extract;

[0041] (2) Concentrate the extract until it has no alcohol taste, and then add petroleum ether, dichloromethane, ethyl acetate, and water to extract the extracts to obtain the extracts;

[0042] (3) The water extraction fraction obtained in step (2) was dissolved in methanol, separated by DAION HP20 macroporous adsorption resin, and eluted with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 90% ethanol aqueous solution in sequence to obtain corresponding fractions;

[0043] (4) taking the fraction eluted with 30% ethanol aqueous solution in step (3), dissolving it with methanol, dry-mixing it with silica gel (100-200 mesh), loading it onto a pre-filled silica gel (100-200 mesh) column, performing column chromatography separation, gradient eluting with dichloromethane-methanol (volume ratio of 100:0, 85:1, 50:1, 25:1, 10:1, 5:1, 0:100), and detecting it with high performance liquid chromatography, combining the fractions with similar liquid phase spectra to obtain 15 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0044] (5) The fraction 12 obtained in step (4) was dissolved in methanol and separated by preparative HPLC using acetonitrile-0.1% formic acid aqueous solution (volume ratio 20:80) as the mobile phase. The isocratic elution was performed at a flow rate of 10 ml / min with a retention time of 35 min to obtain the target compound.

[0045] 1. Structural analysis and identification of compounds

[0046] Structural analysis of compounds: mainly using spectroscopy techniques, including mass spectrometry, nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR, 2D-NMR) to identify its structure, the specific spectrum is as follows Figure 2~Figure 9 As shown, the spectral data and analysis process are as follows:

[0047] (1) The compound is a white powder. ESI-MS m / z 575.1974[MH] - , whose molecular formula is C 26 H 32 N4O 11 , the calculated unsaturation is 13.

[0048] Comprehensive analysis 1 H-NMR, 13 C-NMR shows that the compound has a glucose carbon signal ( d c102.4, d c 73.5, d c 76.8, d c 70.1, d c76.8, d c 61.1), 4 methylene groups ( d c 42.9, d c 36.8, d c34.0, d c 31.2), 6 methylene groups ( d c 82.6, d c 79.3, d c 49.1, d c 45.6, d c 43.5, d c 43.2), 4 carbonyl groups ( d c175.6, d c152.6, d c149.9, d c140.5), 3 pairs of unsaturated double bond carbons ( d c 150.3, d c 149.0, d c129.8, d c 112.9, d c 110.9, d c 97.5). 1 sugar terminal hydrogen signal [ d H : 4.29, d, 1H;], 2 oxymethylene hydrogen signals [ dH : 4.47, t, 1H; 4.17, t, 1H]. Then, the planar structure of the compound was determined by combining two-dimensional NMR spectroscopy data such as HMBC, DEPT and HSQC spectra. The structural formula is as follows: Figure 1 As shown, the compound was named Ainslinine B.

[0049] (2) The spectral data of the compound are as follows:

[0050] 1 H NMR (700 MHz, DMSO- d 6 ) d H 5.38 (1H, s, H a -15),5.16 (1H, s, H b -15),4.86 (1H, s, H a -14), 4.80 (1H, s, H b -14), 4.47 (1H, t, J =7.70, 15.3 Hz, H-3),4.29 (1H, d, J =7.9, H-1′′), 4.17 (1H, t, J =9.7, 19.4 Hz, H-6), 3.98 (2H, m, H-13), 3.69 (1H, d, J = 11.8 Hz, H a -6′′), 3.44 (1H, dd, J = 5.9, 11.8 Hz, H b -6′′),2.97~3.17 (4H, m, H-2′′~ H-5′′), 2.97 (1H, m, H-11), 2.84 (1H, m, H-1),2.68(1H, t, J =9.6, 19.1 Hz, H-5),2.23 (1H, m, H-7), 2.33 (1H, m, H a -9), 2.20, (1H,m, H a -2), 1.91 (1H, m, H b -9), 1.73 (1H, m, H b -2), 1.71 (1H, m, H b -8), 1.35(1H,m, H a -8).

[0051] 13 C NMR (176 MHz, DMSO- d 6 ) d C 175.6 (C-12), 152.6 (C-6′), 150.3 (C-4), 149.9 (C-2′), 149.0 (C-10), 140.5 (C-8′), 129.8 (C-4′), 112.9 (C-14), 110.9 (C-15), 102.4 (C-1′′), 97.5 (C-5′), 82.6 (C-6), 79.3(C-3), 76.8 (C-3′′), 76.8(C-5′′), 73.5 (C-2′′),70.1(C-4′′),61.1(C-6′′),49.1(C-5),45.6(C-11),43.5(C-7),43.2(C-1),42.9(C-13),36.8(C-2),34.0(C-9),31.2(C-8).

[0052] 2. Experimental study of drug activity

[0053] 1. Experimental Materials and Animals

[0054] 1. Drugs and reagents

[0055] The compound prepared in Example 1 (self-extracted, purity above 98%); F / 12K high glucose medium (Wuhan Prosai Life Science Technology Co., Ltd.), CCK-8 cell proliferation and toxicity detection kit (Beijing Solebold Technology Co., Ltd.), A549 human non-small cell lung cancer cells (Wuhan Prosai Life Science Technology Co., Ltd.), fetal bovine serum (Wuhan Prosai Life Science Technology Co., Ltd.), PBS buffer (Beijing Solebold Technology Co., Ltd.), crystal violet (Shanghai Biotech Co., Ltd.).

[0056] 2. Experimental instruments

[0057] Inverted electron microscope (OLYMPUS, Japan: CKX41), microplate reader (Molecular Devices, USA: SpectraMax i3), MCV-B161S (T) clean bench (SANYO, Japan), MCO-20ACI CO2 constant temperature incubator (Thermo, USA), BS125S 1 / 10,000 balance (Mettler Toledo, Switzerland), TDL-5C desktop microcentrifuge (Eppendorf, Germany).

[0058] 2. Experimental methods

[0059] 1. Cytotoxicity test of the compound prepared in Example 1 (CCK8 method)

[0060] First, add the cell suspension containing A549 cells into the culture flask, and then add 10 mL of F / 12K medium containing 10% fetal bovine serum prepared in advance. Culture in an incubator at 37°C and 5% CO2, perform sterile operations throughout the process, and closely observe cell morphology and growth every day. After the cell density is qualified under the microscope, take A549 cells in the logarithmic growth phase and incubate at 5×10 3 The cells were inoculated at a density of 1.5 cells / well in a 96-well plate and administered after 24 hours of culture, namely, a blank group and a drug-administered group (50µM, 100µM, and 100µM compound). After 24 hours of culture, the culture medium was discarded, and 100µl of F / 12K solution containing 10% CCK-8 was added to each well. After 2 hours of culture, the absorbance was detected at a wavelength of 450 nm on a microplate reader and the survival rate of A549 cells was calculated.

[0061] 2. Cloning experiment of the compound obtained in Example 1

[0062] First, add the cell suspension containing A549 cells into the culture flask, and then add 10 mL F / 12K medium containing 10% fetal bovine serum prepared in advance. Cultivate in an incubator at 37 ℃ and 5% CO2, operate aseptically throughout the process, and closely observe the changes in cell morphology and growth every day. After observing that the cell density is qualified under the microscope, take the A549 cells in the logarithmic growth phase and inoculate them in a 6-well plate at a density of 600 cells / well. After culturing for 24 hours, the drugs are administered on 1 day, 3 days, 6 days, and 9 days, respectively, which are the blank group and the drug group (50µM, 100µM, and 200µM compound); replace fresh culture medium or drug-containing culture medium every 2-3 days, continue to culture for two weeks, continue to observe the cell morphology, and terminate the culture when visible clones appear in the culture dish. Discard the culture medium, carefully rinse twice with PBS, and add 1mL of 4% paraformaldehyde (PFA) to fix the cells for 30 minutes. After discarding PFA, 1 mL of 0.1% crystal violet was added to each well for staining for 30 min. The staining solution was washed away with ultrapure water. After the 6-well plate was dried, it was photographed and the clone formation rate was calculated.

[0063] (III) Experimental results

[0064] 1. The compound prepared in Example 1 was tested for A549 cell toxicity at concentrations of 50 μM, 100 μM, and 200 μM, and the results are shown in Table 1. The results show that the prepared compound has a significant growth inhibitory effect on A549 cells.

[0065] Table 1 Effects of compounds on the viability of A549 cells

[0066]

[0067] 2. Cell clone formation assay was used to detect the effect of the compound on tumor cell proliferation. The results are shown in Figure 10 and Table 2. As the concentration increases, the clone formation rate of human non-small cell lung cancer cells (A549) becomes lower, that is, the inhibitory effect of the compound of the present invention on the proliferation performance of human non-small cell lung cancer cells (A549) becomes more obvious.

[0068] Table 2 Effects of compounds on the number of A549 cell clones

[0069]

[0070] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sesquiterpenoid compound in Herba Lycopodii, characterized in that: The chemical structural formula of the sesquiterpenoid compound is as follows: 。 2. A method for preparing a sesquiterpenoid compound in the apricot-scented rabbit ear wind according to claim 1, characterized in that: The following steps are involved: (1) Take the whole herb of Psoralea corylifolia and add ethanol solution for extraction to obtain an extract; (2) The extract is concentrated until there is no alcohol taste, and petroleum ether, dichloromethane, ethyl acetate, and water are added to extract to obtain various extraction parts; (3) dissolving the water extraction fraction obtained in step (2) with methanol, separating it with a macroporous adsorption resin, and eluting it with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 90% ethanol aqueous solution in sequence to obtain corresponding fractions; (4) Take the fraction eluted with 30% ethanol aqueous solution in step (3), dissolve it with methanol, mix it with silica gel dry method, load it into a pre-filled silica gel column, separate it by column chromatography, and use dichloromethane-methanol gradient elution with volume ratio of 100:0, 85:1, 50:1, 25:1, 10:1, 5:1, 0:100 to obtain 15 fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; (5) The fraction 12 obtained in step (4) was dissolved in methanol and separated by preparative high performance liquid chromatography using acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 20:80 as the mobile phase with a retention time of 35 min to obtain the target compound.

3. The method for preparing a sesquiterpenoid compound in the apricot-scented rabbit ear wind according to claim 2, characterized in that: In step (3), the macroporous adsorption resin is DAION HP20 resin.

4. The method for preparing a sesquiterpenoid compound in the apricot-scented rabbit ear wind according to claim 2, characterized in that: In step (4), the silica gel used for sample mixing is 100-200 mesh, and the silica gel used in the silica gel column is 100-200 mesh.

5. The method for preparing a sesquiterpenoid compound in the apricot-scented rabbit ear wind according to claim 2, characterized in that: In step (5), the elution flow rate is 10 ml / min.

6. Use of the sesquiterpenoid compound according to claim 1 in the preparation of anti-lung cancer drugs.

7. A pharmaceutical composition, characterized in that: The invention comprises the sesquiterpenoid compound as claimed in claim 1.

8. A pharmaceutical preparation, characterized in that: The invention comprises a therapeutically effective amount of the sesquiterpenoid compound according to claim 1 and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical preparation according to claim 8, characterized in that: The pharmaceutical preparation is an oral preparation or an injection preparation.

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