A guaiacyl sesquiterpenoid compound and its preparation method and application

Abstract

The present application belongs to the field of medical technology, and discloses a guaiacyl sesquiterpenoid compound and its preparation method and application. Take Xingxiang Rabbit Ear Wind for alcohol extraction, and extract with petroleum ether, dichloromethane, ethyl acetate and water respectively. The water extraction part is first separated by resin, and then separated by silica gel column chromatography, ODS column 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 guaiacyl sesquiterpenoid compound can be separated and obtained. Experiments show that the compound of the present invention can effectively inhibit the expression of ACTA2, COL1A1, VIM and FN1 genes in the TGF-β-induced BEAS-2 pulmonary fibrosis cell model, has anti-pulmonary fibrosis effect, and can be used to prepare anti-pulmonary fibrosis drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a guaiacyl sesquiterpenoid compound and a preparation method and application thereof. Background Art

[0002] Pulmonary fibrosis is a lung disease characterized by damage to lung tissue and subsequent excessive proliferation of fibrous tissue, which gradually leads to loss of lung function. The occurrence of pulmonary fibrosis may be related to a variety of factors, including occupational exposure (such as long-term exposure to dust and chemicals), smoking, radiation therapy, use of specific drugs (such as chemotherapeutic drugs and immunosuppressants), and connective tissue diseases. Although the exact etiology and pathogenesis of pulmonary fibrosis have not been fully elucidated, it is generally believed that it involves damage to alveolar epithelial cells, abnormal repair processes, activation of fibroblasts, and abnormal deposition of extracellular matrix, which together promote the destruction of lung structure and loss of function. At present, the treatment of pulmonary fibrosis mainly focuses on relieving symptoms, delaying disease progression, and improving quality of life. Existing anti-fibrotic drugs, such as pirfenidone and nintedanib, have been shown to slow the rate of decline in lung function. With the increasing demand for drugs for the treatment of pulmonary fibrosis, the role and mechanism of Chinese medicine monomers in the treatment of pulmonary fibrosis have gradually attracted attention. Studies have shown that Chinese medicine monomers exert their anti-inflammatory and antioxidant effects by acting on the TGFβ / Smad pathway, NF-κB signaling pathway, PI3K / Akt signaling pathway, etc., reducing the deposition of extracellular matrix and the production of inflammatory factors, thereby producing a positive therapeutic effect on pulmonary fibrosis. The multi-target, multi-pathway, and multi-pathway characteristics of Chinese medicine monomers have made them a hot topic in current drug research, providing broad prospects for the development of new anti-pulmonary fibrosis drugs from Chinese medicine.

[0003] Apricot scented rabbit ear wind Ainsliaea fragrans Champ . It is a perennial herb of the genus Rhizoma in the Asteraceae family. It 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 Psoralea corylifolia, provide a guaiacane-type sesquiterpenoid compound and a preparation method and application thereof, and the research finds that the guaiacane-type sesquiterpenoid compound of the present invention has a good anti-pulmonary fibrosis effect.

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

[0006] A guaiacyl sesquiterpenoid compound named Ainslinine A has the following chemical structure:

[0007] .

[0008] The present invention also provides a method for preparing the guaiacyl 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) 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;

[0011] (3) taking the water extraction part obtained in step (2), dissolving it with methanol, separating it with DAION HP20 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;

[0012] (4) taking the fraction obtained from the elution portion of the 30% ethanol aqueous solution in step (3), dissolving it with methanol, dry-mixing it with silica gel (100-200 mesh), loading it onto a packed silica gel (100-200 mesh) column, performing column chromatography separation, gradient eluting with dichloromethane-methanol (volume ratio 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 11 obtained in step (4) was dissolved in methanol, separated using an ODS column, and gradient eluted with methanol-water (volume ratio 10:90, 30:70, 50:50, 70:30, 90:10) to obtain five fractions a, b, c, d, and e;

[0014] (6) The fraction b obtained in step (5) was dissolved in methanol and separated by preparative HPLC using acetonitrile-0.1% formic acid aqueous solution (volume ratio 22:78) as the mobile phase. The isocratic elution was performed at a flow rate of 10 ml / min with a retention time of 49 min to obtain the target compound.

[0015] 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 60-90°C.

[0016] Preferably, the ethanol solution used in step (1) is an ethanol aqueous solution with a volume concentration of 5% to 95%, more preferably a 70% 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 12 times.

[0017] 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.

[0018] The present invention also provides the use of the guaiacyl sesquiterpenoid compound in the preparation of an anti-pulmonary fibrosis drug.

[0019] The present invention also provides a pharmaceutical composition comprising the guaiacane-type sesquiterpenoid compound.

[0020] The present invention also provides a pharmaceutical preparation, comprising a therapeutically effective amount of the guaiacyl sesquiterpenoid compound and a pharmaceutically acceptable carrier or excipient.

[0021] 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.

[0022] Preferably, the oral preparation is a tablet, a capsule, a granule, a fat emulsion, a microcapsule, or a pill.

[0023] Preferably, the injection preparation is an injection solution or a powder injection.

[0024] The beneficial effects of the present invention are:

[0025] The preparation method of the compound of the present invention is simple to operate and can separate and obtain the guaiacane-type sesquiterpenoid compound.

[0026] Experiments show that the compound of the present invention can effectively inhibit the expression of SMA, FN1, Vim and Collal genes of BEAS-2B cells induced by TGF-β, has a good anti-pulmonary fibrosis effect, and can be used for the preparation of anti-pulmonary fibrosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the chemical structural formula of the compound of the present invention;

[0028] Figure 2 is the mass spectrum of the compound of the present invention;

[0029] Figure 3 The compounds of the present invention 1 H-NMR spectrum;

[0030] Figure 4 The compounds of the present invention 13 C-NMR spectrum;

[0031] Figure 5 is the DEPT spectrum of the compound of the present invention;

[0032] Figure 6 is the HSQC spectrum of the compound of the present invention;

[0033] Figure 7 is the HMBC spectrum of the compound of the present invention;

[0034] Figure 8 The compounds of the present invention 1 H- 1 H COSY spectrum;

[0035] Figure 9 It is the NOESY spectrum of the compound of the present invention. 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 guaiacyl sesquiterpenoid compounds in Apricot-scented Rabbit Ear Wind, the steps are as follows:

[0040] (1) Take the whole herb of Psoralea corylifolia, add 12 times the volume of 70% ethanol aqueous solution, reflux and extract twice, each time for 2 hours, to obtain an extract;

[0041] (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;

[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 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) Dissolve the fraction 11 in step (4) in methanol, separate using an ODS column, and gradient elute using methanol-water (volume ratio 10:90, 30:70, 50:50, 70:30, 90:10) to obtain five fractions a, b, c, d, and e;

[0045] (6) The fraction b obtained in step (5) was dissolved in methanol and separated by preparative HPLC using acetonitrile-0.1% formic acid aqueous solution (volume ratio 22:78) as the mobile phase. The isocratic elution was performed at a flow rate of 10 ml / min with a retention time of 49 min to obtain the target compound.

[0046] 1. Structural analysis and identification of compounds

[0047] 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:

[0048] (1) White powder, ESI-MS m / z 536.2159 [MH] - The compound formula is C 26 H 35 NO 11 , the calculated unsaturation is 10.

[0049] Comprehensive analysis 1 H-NMR, 13 C-NMR shows that the compound has a glucose carbon signal ( d c102.9, d c 73.6, d c 76.8, d c 70.1, d c 76.9, d c 61.1), 6 methylene groups ( d c 39.4, d c 36.7, d c35.9, d c 31.6, d c 29.0, d c 23.0), 7 methylene groups ( d c 83.1, d c 79.3, d c 60.8, d c 48.2, d c45.3, d c 44.8, d c 42.6, ), 3 carbonyl groups ( d c 176.6, d c 175.5, d c 174.0), 2 pairs of unsaturated double bond carbons ( d c150.4, d c 149.4, d c 112.6, d c 109.8). 1 sugar terminal hydrogen signal [ d H : 4.27, d, 1H;], 2 oxymethylene hydrogen signals [ d H : 4.45, t, 1H; 4.05, 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 AinslinineA.

[0050] (2) The spectral data of the compound are as follows:

[0051] 1 H-NMR (700 MHz, DMSO- d 6 ) d H 5.38 (1H, d, J = 2.0 Hz, H a -15),5.12 (1H, d, J = 2.0 Hz, H b -15), 4.85, (2H, s, H-14), 4.45 (1H, t, J = 7.6, 15.2 Hz, H-3),4.27 (1H, d, J = 7.8 Hz, H-1′′),4.05 (1H, t, J= 9.7, 19.4 Hz, H-6), 3.98 (1H,d, J = 6.5 Hz, H-2′), 3.78 (2H, dd, J = 5.1 14.2 Hz, H-13),3.68 (1H, dd, J = 2.1,11.9 Hz, H a -6′′), 3.43 (1H, dd, J = 5.9, 11.9 Hz, H b -6′′),3.00~3.15 (4H, m, H-2′′~ H-5′′), 2.83 (1H, dd, J = 8.3, 17.0 Hz, H-1), 2.74 (1H, m, H-5), 2.67 (1H,m, H-11),2.38 (1H, m, H a -9), 2.27 (2H, m, H-4′), 2.25 (1H, m, H a -2), 2.23 (1H,m, H-7),2.05 (1H, m, H a -3′), 2.05 (1H, m, H a -8), 1.91 (1H, m, H b -3′), 1.90(1H, m, H b -9),1.73 (1H, m, H b -2), 1.28(1H, m, H b -8)。

[0052] 13 C-NMR (176 MHz, DMSO- d 6 ) d C176.6 (C-12), 175.5 (C-6′), 174.0 (C-5′), 150.4 (C-4), 149.4 (C-10), 112.6 (C-14), 109.8 (C-15), 102.9 (C-1′′), 83.1(C-6), 79.3 (C-3), 76.9 (C-3′′), 76.8 (C-5′′), 73.6 (C-2′′), 70.1 (C-4′′), 61.1 (C-6′′), 60.8 (C-2′), 48.2 (C-5), 45.3 (C-11), 44.8 (C-7), 42.6(C-1),39.4 (C-13), 36.7 (C-2), 35.9 (C-9), 31.6 (C-8), 29.0 (C-4′), 23.0 (C-3′).

[0053] 2. Experimental study on drug activity

[0054] 1. Experimental Materials and Animals

[0055] 1. Drugs and reagents

[0056] The compound prepared in Example 1 (self-extracted, purity above 98%); DMEM high glucose medium (Beijing Solebow Technology Co., Ltd.), BEAS-2B cells (Haixing Biotechnology Co., Ltd.), fetal bovine serum (Beijing Solebow Technology Co., Ltd.), TGF-β (Wuhan Sanying Biotechnology Co., Ltd.), SteadyPure rapid RNA extraction kit (Hunan Aikerui Biotechnology Co., Ltd.), HifairAdvanceFast 1st Strand cDNA Synthesis SuperMix for qPCR (Yishen Biotechnology (Shanghai) Co., Ltd.), SYBR Green Premix Pro Taq HS qPCR Kit (Hunan Aikerui Biotechnology Co., Ltd.), mRNA primer (Hunan Aikerui Biotechnology Co., Ltd.).

[0057] 2. Experimental instruments

[0058] 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), PQTOWER3G fluorescence quantitative PCR instrument (Jena Analytical Instruments AG, Germany).

[0059] 2. Experimental methods

[0060] Anti-fibrosis study of the compound obtained in Example 1

[0061] First, add 5 mL of high-glucose DMEM medium containing 10% fetal bovine serum into a T75 culture flask, and then add BEAS-2B cell suspension. Place in an incubator at 37°C and 5% CO2, and culture aseptically throughout the process. Observe cell morphology and growth until the cell density is appropriate. Then, take BEAS-2B cells in the logarithmic growth phase and inoculate them into 24 wells, with 1×10 cells per well. 5 cells, cultured to a density of about 80%, and then administered. The cells were divided into a blank group, a model group, and a drug group. The drug group was added with 1 mL of medium containing 200 μM of the compound, and the blank group was added with an equal amount of medium. After 2 h, except for the blank group, the remaining groups were added with an appropriate amount of TGF-β to make the final concentration 10 ng / ml. After 72 h of continuous culture, the cell RNA was extracted using the SteadyPure fast RNA extraction kit, and then the RNA was reverse transcribed into cDNA using the HifairAdvanceFast 1st Strand cDNA Synthesis SuperMix forqPCR. The SYBR Green PremixPro Taq HS qPCR Kit reagent was used for quantification, the qPCR reaction system was configured, and the PCR reaction program was set. After the reaction was completed, the data was copied to an Excel spreadsheet for data processing, and the data were statistically analyzed using Graghpad Prism 8.

[0062] (III) Experimental results

[0063] The effects of the compound prepared in Example 1 on the expression levels of ACTA2, FN1, VIM and COLLAL genes in BEAS-2B cells induced by TGF-β are shown in Table 1.

[0064] Table 1

[0065]

[0066] The results in Table 1 show that the prepared compound has an inhibitory effect on the expression of ACTA2, FN1, VIM and COLLAL genes in the TGF-β-induced BEAS-2B cell pulmonary fibrosis model, suggesting that it has an anti-pulmonary fibrosis effect.

[0067] 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 guaiacyl sesquiterpenoid compound, characterized in that: The chemical structural formula of the guaiacyl sesquiterpenoid compound is as follows: 。 2. A method for preparing the guaiacyl sesquiterpenoid compound 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) Concentrating the extract, and respectively adding petroleum ether, dichloromethane, ethyl acetate, and water for extraction to obtain various extraction fractions; (3) taking the water extraction part obtained in step (2), dissolving it 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 obtained from the elution portion of the 30% ethanol aqueous solution in step (3), dissolve it in methanol, mix it with silica gel dry method, load it into a packed silica gel column, separate it by column chromatography, and use dichloromethane-methanol gradient elution with a 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 11 obtained in step (4) was dissolved in methanol, separated using an ODS column, and eluted with a methanol-water gradient with a volume ratio of 10:90, 30:70, 50:50, 70:30, and 90:10 to obtain five fractions a, b, c, d, and e; (6) The fraction b obtained in step (5) was dissolved in methanol and separated by preparative HPLC using acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 22:78 as the mobile phase for elution with a retention time of 49 min to obtain the target compound.

3. The method for preparing the guaiacyl sesquiterpenoid compound according to claim 2, characterized in that: In step (3), the macroporous adsorption resin is DAION HP20 resin.

4. The method for preparing the guaiacyl sesquiterpenoid compound 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 the guaiacyl sesquiterpenoid compound according to claim 2, characterized in that: In step (5), the elution flow rate is 10 ml / min.

6. Use of the guaiacyl sesquiterpenoid compound according to claim 1 in the preparation of an anti-pulmonary fibrosis drug.

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