Application of formononetin derivative in preparation of medicine for preventing and treating acute lung injury

Abstract

The invention relates to a novel application of a formononetin derivative, namely succinyl formononetin (formonone-7-O-(6-O-succinyl)-D-glucoside) in medicine, in particular to an application of the formononetin derivative in preparation of a medicine for treating and preventing acute lung injury. Pharmacological experiment results prove that compared with prototype formononetin, the formononetin derivative, namely succinyl formononin, can relieve the occurrence and development of lung injury of model animals to different degrees in different indexes; the compound can relieve the damage of inflammation to lung epithelial cells in vitro, and can be used for preparing the medicine for treating lung injury, especially the medicine for treating acute lung injury.

Description

Technical Field

[0001] The present invention relates to a new use of formononetin derivatives in medicine, specifically to the application of formononetin derivatives in the preparation of drugs for treating and preventing acute lung injury. Background Art

[0002] Acute lung injury is an acute and diffuse pulmonary inflammatory disease caused by various etiologies, manifested as damage to the alveolar-capillary membrane, pulmonary edema, gas exchange disorders, and refractory hypoxemia, leading to a sharp decline in lung function and may further develop into acute respiratory distress syndrome, with extremely high morbidity and mortality, having a great impact on public health. Currently, for patients with acute lung injury, mainly through protective mechanical ventilation, anti-inflammatory drugs and glucocorticoids such as dexamethasone, pulmonary vasodilators, statins and other drugs for intervention. Although it can play a certain therapeutic effect on acute lung injury, there are a series of side effects to varying degrees.

[0003] Flavonoids are products of plant secondary metabolism, with a wide variety of types and diverse structures, showing rich pharmacological activities, and can play an important role in the prevention and treatment of cardiovascular and respiratory diseases. As one of the representative flavonoids, formononetin exhibits various pharmacological activities, can regulate airway inflammation by inhibiting airway inflammation, and can prevent respiratory diseases. However, the low water solubility of formononetin limits its use to a certain extent. And a formononetin derivative, succinyl formononetin glycoside, has high water solubility and is more low-toxic and safe. At the same time, succinyl formononetin glycoside can achieve effective treatment of cardiovascular diseases and other functions, while the effect of formononetin derivative - succinyl formononetin glycoside drug on preventing and treating acute lung injury compared with formononetin is the key of the present invention. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of formononetin derivatives in medicine, specifically the application of formononetin derivatives in the preparation of drugs for treating and preventing acute lung injury.

[0005] The purpose of the present invention is achieved by the following technical solutions: The existing compound succinyl formononetin glycoside, which is formononetin-7-O-(6-O-succinyl)-D-glucoside, and the obtained succinyl formononetin glycoside structure is shown in Formula 1:

[0006] Formula 1:

[0007]

[0008] The present invention provides the application of a formononetin derivative in the preparation of drugs for treating and preventing acute lung injury.

[0009] Preferably, the acute lung injury includes hemoptysis, respiratory distress, respiratory failure, hypoxemia, and hypercapnia.

[0010] Preferably, the formononetin derivative in the drug is used as the sole active ingredient.

[0011] Preferably, the drug comprises a formononetin derivative, a pharmaceutically acceptable salt, ester, hydrate of the formononetin derivative or a combination thereof, and excipients.

[0012] Preferably, the formononetin derivative salt refers to a salt formed by a formononetin derivative and hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, malonic acid, succinic acid, glutaric acid, maleic acid, an alkyl or aryl sulfonic acid.

[0013] Preferably, the effective treatment amount of succinyl formononetin is 10 mg / kg - 40 mg / kg.

[0014] Preferably, the dosage form of the drug is selected from tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, medicinal wines, tinctures, extracts, films or combinations thereof.

[0015] Preferably, the administration methods of the drug include oral administration, injection, implantation, external use, spraying, inhalation or combinations thereof.

[0016] Beneficial effects

[0017] 1. The succinyl formononetin of the present invention inhibits pulmonary inflammation and pulmonary pathological changes, and reduces the levels of related cytokines in lung tissues and bronchoalveolar lavage fluid.

[0018] 2. The succinyl formononetin of the present invention protects lung epithelial cells from inflammatory damage, promotes migration and inhibits apoptosis.

[0019] 3. The experimental results of the water solubility analysis of formononetin, formononetin derivatives - formononetin glycoside, and succinyl formononetin of the present invention show that:

[0020] The water solubility of succinyl formononetin is significantly improved compared with that of formononetin. The results show that the water solubility of succinyl formononetin is increased by about 822 times compared with that of formononetin.

[0021] 4. The experimental results of the present invention on mice show that:

[0022] (1) The effect of formononetin derivative - succinyl formononetin glycoside in improving the degree of lung injury in mice with LPS-induced acute lung injury model is increased to 200% compared with formononetin, and the effect of formononetin glycoside in improving the degree of lung injury in mice with LPS-induced acute lung injury model is increased to 130% compared with formononetin, which is statistically significant;

[0023] (2) The effect of formononetin derivative - succinyl formononetin glycoside in improving the lung pathological changes and reducing the lung pathological score in model mice is increased to 250% compared with formononetin, and the effect of formononetin glycoside in improving the lung pathological changes and reducing the lung pathological score in model mice is increased to 130% compared with formononetin, which is statistically significant;

[0024] (3) The effect of formononetin derivative - succinyl formononetin glycoside in reducing cytokines IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of mice is increased to 200%-250% compared with formononetin, and the effect of formononetin glycoside in reducing cytokines IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of mice is increased to 130%-180% compared with formononetin, which is statistically significant.

[0025] 5. The in vitro lung epithelial cell culture experiment of the present invention shows that:

[0026] (1) The formononetin derivative - succinyl formononetin glycoside has no toxic effect on lung epithelial cells;

[0027] (2) The effect of formononetin derivative - succinyl formononetin glycoside in inhibiting the expression of IL-1β, IL-6, and TNF-α in lung epithelial cells after LPS stimulation is increased to 250%-300% compared with formononetin, and the effect of formononetin glycoside in inhibiting the expression of IL-1β, IL-6, and TNF-α in lung epithelial cells after LPS stimulation is increased to 130%-200% compared with formononetin, which is statistically significant;

[0028] (3) The effect of formononetin derivative - succinyl formononetin glycoside in promoting the migration of lung epithelial cells after LPS stimulation is increased to 140% compared with formononetin, and the effect of formononetin glycoside in promoting the migration of lung epithelial cells after LPS stimulation is increased to 110% compared with formononetin, which is statistically significant;

[0029] (4) The effect of formononetin derivative - succinyl formononetin glycoside in inhibiting the apoptosis of lung epithelial cells after LPS stimulation is increased to 120% compared with formononetin, and the effect of formononetin glycoside in promoting the migration of lung epithelial cells after LPS stimulation is increased to 105% compared with formononetin, which is statistically significant. Description of the Drawings

[0030] Figure 1 is the formononetin content in licorice residue;

[0031] Figure 2Comparison of dry weight / wet weight of the lungs of mice in each experimental group;

[0032] Figure 3 Comparison of lung pathology of mice in each experimental group (X200);

[0033] Figure 4 Changes in the levels of IL-4, IL-5, and IL-13 in the airway lavage fluid of mice in each experimental group;

[0034] Figure 5 Effect of formononetin derivatives at different doses on cell proliferation.

[0035] Figure 6 Effect of formononetin, formononetin glycoside, and succinyl formononetin glycoside on the expression of cell inflammatory factors IL-1β, IL-6, and TNF-α after LPS stimulation.

[0036] Figure 7 Effect of formononetin, formononetin glycoside, and succinyl formononetin glycoside on cell migration after LPS stimulation (X100).

[0037] Figure 8 Effect of formononetin, formononetin glycoside, and succinyl formononetin glycoside on cell apoptosis after LPS stimulation. Detailed implementation manners

[0038] To further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0039] Example 1 Efficient acquisition of formononetin compounds from licorice residues

[0040] Extracting flavonoid compounds from Chinese medicine residues includes the following steps:

[0041] Place 5 grams of licorice residues in a 250 mL conical flask, add 3 g / L of (NH4)2SO4, 0.5 g / L of urea, 2 g / L of KH2PO4, 0.5 g / L of MgSO4, 0.1% metal ions (FeSO4·7H2O, 0.005 g / L, MnSO4·H2O 0.0016 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.002 g / L) and 100 mL of pure water, sterilize in a 121°C autoclave for 20 minutes; after cooling to 28°C, inoculate Penicillium Oxalicum Z2 (CCTCC NO: M 20241501) preserved in the China Center for Type Culture Collection (inoculation amount 1 wt%, and the concentration of the strain after inoculation is 1×10 6(CFU / mL), fermentation (conditions: temperature 28 °C, rotation speed 200 r / min, fermentation time 4 days). After fermentation, the temperature was raised to 55 °C for in-situ enzymatic hydrolysis for 10 hours, and then the temperature was raised to 90 °C and kept warm for 15 minutes. After enzymatic hydrolysis, it was cooled to room temperature and centrifuged at 5000 rpm, and filtered through a 200-mesh gauze to obtain the licorice residue extract.

[0042] Using a preparative liquid chromatography system equipped with XB-C18 (5-μm particle size, 250 mm in length, 10 mm in inner diameter), separation was carried out at a flow rate of 3 mL / min. A mobile phase of methanol and formic acid water was used for isocratic elution at a mass ratio of 60:40. The retention time of formononetin was approximately 14.56 - 15.12 min, and the content accounted for approximately 30% of the licorice residue extract. The experimental results are shown in Figure 1 . Subsequently, the fraction was concentrated to a small volume at 40 °C using a rotary evaporator, and a solid powder-like compound was obtained by freeze-drying technology. The purity of the components was detected by HPLC-UV to ensure that the purity reached over 90%.

[0043] The compound isolated from the extract was used 1 1H-NMR and other means, combined with the references, to determine its structure. The obtained formononetin structure is shown in Formula 2;

[0044] Formula 2:

[0045]

[0046] Formononetin: white amorphous powder. 1 1H-NMR (400 MHz, DMSO-d6) δ ppm 3.79 (S, 3H) 6.87 (d, J = 2.08 Hz, 1H) 6.94 (dd, J = 8.80, 2.20 Hz, 1H) 6.99 (m, 8.80 Hz, 2H) 7.97 (d, J = 8.68 Hz, 1H) 8.34 (s, 1H) 10.80 (br.s., 1H)

[0047] In this example, a total of 0.29 g of formononetin solid powder was obtained from 5 g of licorice residue.

[0048] Example 2 Obtaining formononetin glycoside and succinyl formononetin glycoside by biotransformation

[0049] Inoculate the fermentation broth of Bacillus amyloliquefaciens FJ18 into a seed medium: yeast extract 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L, pH 7.0, and culture at 30 °C and 200 rpm for 12 hours. The components and contents of the expansion medium and the fermentation medium are both: sucrose 20 g / L, yeast powder 15 g / L, KH2PO4 1.0 g / L, CaCl2 0.8 g / L. Adjust the pH to 8.0 with NaOH. Inoculate the seed liquid into the expansion medium and the fermentation medium at 0.5% (v / v), and culture at 30 °C and 200 rpm for 12 hours. Collect the thallus cells by centrifugation at 10000 rpm for 15 minutes, and wash them with physiological saline 1 - 2 times to obtain the resting cells of Bacillus amyloliquefaciens FJ18.

[0050] Filter the thallus cell fermentation broth to obtain wet thalli. Prepare a raw material solution, namely the reaction solution, with dimethyl sulfoxide, formononetin, sucrose, and phosphate buffer. The proportion of the organic solvent dimethyl sulfoxide in the reaction solution is 20% (v / v), formononetin 0.5 g / L, the molar concentration of the phosphate buffer is 150 mmol / L, the pH of the phosphate buffer is 8.0, and the sucrose concentration is 50 g / L. Disperse the obtained wet thalli in the reaction solution, add it to a reactor, and culture at 30 °C and 200 rpm for 24 h. Then, centrifuge at 10000 rpm for 10 minutes to obtain the supernatant of the reaction solution. The conversion rate of formononetin measured by HPLC analysis is 97.2%.

[0051] Separate the product with macroporous resin. Take an appropriate amount of resin, soak it in ethanol for 24 h, and remove the resin fragments and debris. Pack the column wet and wash it with 1 L of ethanol, then wash it with distilled water until there is no alcohol smell; then perform acid-base treatment, that is, pass a 5% (v / v) HCl solution and a 2% (w / w) NaOH solution through the resin column at a flow rate of 2 BV / h respectively, and let it stand for 2 - 4 h, and then wash it with distilled water until the pH is neutral. To avoid the dissolution of the conversion product by DMSO and reduce the sample loading adsorption rate, the conversion solution is diluted with 5 times the volume of deionized water (pH 4.0, adjusted with glacial acetic acid) to DMSO < 2% and then sampled. The sample loading amount is 20 mg / g of wet resin, and the sample loading flow rate is 20 mL / min. Wash the excess unreacted glycosyl donor (sucrose) with 10 times the column bed volume of deionized water (pH 4.0, adjusted with glacial acetic acid) until no sugar can be detected in the eluate by concentrated sulfuric acid, and the flow rate is 20 mL / min. Select methanol plus deionized water as the mobile phase for elution, adjust the volume ratio of methanol and deionized water, and determine the methanol ratio in the eluate at an elution flow rate of 20 mL / min. Concentration and drying: After HPLC detection, combine the eluates, and concentrate them under reduced pressure with a rotary evaporator at a heating temperature of 40 °C. Finally, place the solid in a vacuum drying oven and dry it at 40 °C for 6 h.

[0052] The reaction chemical formula for the preparation of succinosideretin in non-aqueous phase by Bacillus amyloliquefaciens FJ18 is shown in the following formula:

[0053]

[0054] As identified by nuclear magnetic resonance mass spectrometry analysis, from the NMR spectrum, the obtained structure is consistent with the structure of the expected product. The above results confirm that glucosylated formononetin is generated in this reaction, that is

[0055] succinosideretin. The nuclear magnetic resonance spectral data of succinosideretin are as follows:

[0056] 1H-NMR(MeOH-d6,600MHz)δ: 8.39(1H,s, 2-H), 8.07(1H,d,J = 8.9Hz, 5-H), 7.52(2H,d,J = 8.6Hz, 2' and 6'-H), 7.24(1H,d,J = 2.3Hz, 8-H), 7.15(1H,dd,J = 8.9Hz,2.4Hz, 6-H), 7.0(2H,d,J = 8.6Hz, 3' and 5'-H), 5.15(1H,d,J = 7.4Hz, 1”-H), 4.42(1H,dd,J = 11.0Hz, 2.4Hz,6”-HA), 4.04(1H,dd,J = 11.0Hz, 7.2Hz,6”-HB), 3.79(3H,s,-OCH3), 2.47 - 2.57(4H,m, 2”' and 3”'-H).

[0057] 13C NMR(MeOH-d6,600MHz)δ: 174.7(C-4), 173.4(C-4”'), 172.0(C-1”'), 161.2(C-7), 159.1(C-4'), 157.0(C-9), 153.6(C-2), 130.1(C-2',6'), 127.0(C-5), 124.0(C-3), 123.4(C-1'), 118.6(C-10), 115.6(C-6), 113.7(C-3',5'), 103.5(C-8), 99.8(C-1”), 76.3(C-3”), 74.0(C-5”), 73.1(C-2”), 69.9(C-4”), 63,6(C-6”), 55.2(C-OCH3)28.7(C-2”'), 28.7(C-3”').

[0058] The structure of the obtained product formononetin glycoside is shown in Formula 3:

[0059] Formula 3:

[0060]

[0061] The structure of the obtained product, formononetin succinate, is shown in Formula 1:

[0062]

[0063] Example 3: Water solubility analysis of formononetin, formononetin glycoside, and formononetin succinate

[0064] A standard curve was established on HPLC, and the solubility of saturated aqueous solutions of formononetin, formononetin glycoside, and formononetin succinate was measured at 37 °C respectively. Experimental results: The water solubility of formononetin was 0.017 g / L, the water solubility of formononetin glycoside was 6.106 g / L; the water solubility of formononetin succinate was 13.967 g / L. The results showed that the water solubility of formononetin succinate was increased by about 822 times compared with formononetin.

[0065] Example 4: Improvement effect on the degree of lung injury in mice

[0066] Balb / C mice were randomly divided into 6 groups, with 12 mice in each group: (1) blank control (Ctrl) group; (2) model (Mod) group; (3) dexamethasone (DXM) group; (4) formononetin (F) group; (5) formononetin glycoside (FG) group; (4) formononetin succinate (FMP) group. Mice in the dosing groups were intragastrically administered dexamethasone (5 mg / kg), formononetin group (40 mg / kg), formononetin glycoside group (40 mg / kg), and formononetin succinate group (40 mg / kg) respectively. Mice in the blank control group and the model group were intragastrically administered the corresponding volume of normal saline. After continuous administration for 3 days, a mouse acute lung injury model was established by nasal instillation of 10 mg / kg LPS 1 h after the last administration. 24 h after the last stimulation, the animals were sacrificed, and the middle and lower lobes of the right lung of the mice were taken. The surface moisture was fully blotted dry with filter paper, and the mass was weighed with an analytical balance and recorded as the wet weight; the tissue was placed in a constant temperature drying oven at 60 °C and baked to a constant weight, and the mass of the lung tissue was weighed and recorded as the dry weight. The W / D was calculated according to the following formula. The experimental results are shown in Figure 2 , the degree of lung injury in the model group was significantly increased, showing a significant difference compared with the normal control group (P < 0.01). The degree of lung injury in the formononetin succinate group was reduced, and it was increased to 200% compared with formononetin, showing statistical significance (P > 0.01, P < 0.05). The improvement effect of formononetin glycoside on the degree of lung injury in the LPS-induced acute lung injury model mice was increased to 130% compared with formononetin.

[0067] Example 5: Improvement effect on mouse lung pathological changes and reduction of lung pathological score

[0068] Balb / C mice were randomly divided into 6 groups, with 12 mice in each group: (1) blank control (Ctrl) group; (2) model (Mod) group; (3) dexamethasone (DXM) group; (4) formononetin (F) group; (5) formononetin (FG) group; (4) succinylated formononetin (FMP) group. The mice in the drug groups were gavaged with dexamethasone (5 mg / kg), formononetin (40 mg / kg), formononetin (40 mg / kg), and succinylated formononetin (40 mg / kg), respectively. The mice in the blank control group and model group were gavaged with the corresponding volume of normal saline for 3 consecutive days. 1 hour after the last administration, 10 mg / kg LPS was dripped into the nasal cavity to establish the mouse acute lung injury model. 24 hours after the last stimulation, the animals were killed, and their lungs were removed and fixed with 10% formalin. The sections were embedded in paraffin and sliced ​​with a thickness of about 4-5 μm, and stained with HE. According to the severity of the lesion, semi-quantitative analysis was performed as mild “+”, moderate “++”, severe “+++”, and tissue without lesion was marked as “-”.

[0069] In the model group, the thickness of bronchial smooth muscle increased significantly (+++), and there was moderate exudate in the lumen; a large number of inflammatory cells infiltrated in the tracheal submucosal layer and muscular layer (+++), mainly including eosinophils, lymphocytes, macrophages and a small number of plasma cells. The bronchial ciliary epithelium was partially detached, the cilia adhered and collapsed, degenerated and necrotic, and the airway epithelium was stratified (+++). In the dexamethasone group, the thickness of bronchial smooth muscle increased slightly (+), and there was no exudate in the lumen; a small number of eosinophils, lymphocytes and a small number of plasma cells infiltrated in the tracheal submucosal layer and muscular layer (+). The structure of the pulmonary lobule was generally normal, and the bronchial cilia adhered and collapsed slightly and degenerated (+). In the formononetin group, the thickness of the bronchial smooth muscle increased moderately (++), and there was moderate exudate in the lumen; inflammatory cell infiltration was seen in the tracheal submucosa and muscular layer (++), mainly including eosinophils, lymphocytes, macrophages, and a small number of plasma cells. The bronchial ciliary epithelium was partially sloughed, the cilia adhered and fell, degenerated, and the airway epithelium was stratified (++). In the formononetin group, the thickness of the bronchial smooth muscle increased slightly (+), and there was exudate in the lumen; inflammatory cell infiltration was seen in the tracheal submucosa and muscular layer (+), mainly including eosinophils, lymphocytes, macrophages, and a small number of plasma cells. The bronchial ciliary epithelium was partially sloughed, the cilia adhered and fell, degenerated, and necrotic (++), and the airway epithelium was stratified (++). In the succinylated formononetin group, the thickness of the lung bronchial smooth muscle increased slightly (+), and there was exudate in the lumen; inflammatory cell infiltration (+) was seen in the tracheal submucosal layer and muscular layer, mainly including eosinophils, lymphocytes, macrophages and a small number of plasma cells. The bronchial ciliary epithelium was partially detached, and the ciliary adhesions collapsed, degenerated, and necrotic (+). The airway epithelium was stratified (+). The lung pathology of mice in each experimental group was compared. Figure 3As shown, the effect of formononetin-7-O-succinate in improving the lung pathological changes and reducing the lung pathological score of model mice was increased to 250% compared with formononetin, and the effect of formononetin glucoside in improving the lung pathological changes and reducing the lung pathological score of model mice was increased to 130% compared with formononetin, which was statistically significant.

[0070] Example 6 Improvement effect on the expression of inflammatory factors IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid

[0071] Balb / C mice were randomly divided into 6 groups with 12 mice in each group: (1) blank control (Ctrl) group; (2) model (Mod) group; (3) dexamethasone (DXM) group; (4) formononetin (F) group; (5) formononetin glucoside (FG) group; (4) formononetin-7-O-succinate (FMP) group. The mice in the drug administration groups were intragastrically administered dexamethasone (5 mg / kg), formononetin group (40 mg / kg), formononetin glucoside group (40 mg / kg), and formononetin-7-O-succinate group (40 mg / kg), respectively. The mice in the blank control group and the model group were intragastrically administered the corresponding volume of normal saline. After continuous administration for 3 days, an acute lung injury model was established by intranasal instillation of 10 mg / kg LPS 1 h after the last administration. 24 h after the last challenge, the animals were sacrificed, the chest cavity was opened, the trachea was exposed, and after ligation of the right bronchus with cotton thread, 0.8 mL of pre-cooled phosphate buffered saline (PBS) was injected into the left lung through the main bronchus using a 1 mL syringe, and the lavage was repeated 3 times. The lavage fluid was collected and stored at -80 °C for later use. The levels of IL-4, IL-5, and IL-13 cytokines were detected by ELISA. The results showed that the expressions of IL-4, IL-5, and IL-13 in the model group were significantly increased, showing significant differences compared with the normal control group (P < 0.01). The expression of IL-4 in the formononetin-7-O-succinate group was decreased, showing statistical significance compared with the formononetin group (P > 0.01, P < 0.05). The expression of IL-5 was significantly decreased, showing significant differences compared with the formononetin group (P < 0.01). The expression of IL-13 was decreased, showing significant differences compared with the formononetin group (P < 0.05). The experimental results are shown in Figure 4 , the effect of formononetin-7-O-succinate in reducing the cytokines IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of mice was increased to 200% - 250% compared with formononetin, and the effect of formononetin glucoside in reducing the cytokines IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of mice was increased to 130% - 180% compared with formononetin, which was statistically significant.

[0072] Example 7 Toxic effect on lung epithelial cells

[0073] Mouse lung epithelial cells MLE-12 were selected for cytotoxicity testing. The cells were digested with trypsin and counted, and the cell concentration was adjusted to 1×10 5cells / mL. MLE-12 cells were seeded in a 96-well plate containing DMEM / F12 high-glucose medium supplemented with 10% fetal bovine serum and then cultured in a cell incubator at 37°C with 5% CO2 for 24 h. Eight concentration groups were set: 0, 1.5, 3, 6.25, 12.5, 25, 50, and 100 μmol / L, with 3 replicates in each group. After 24 h of drug administration, 10 μL of CCK-8 solution was added to each well, and then the cells were further cultured in the incubator at 37°C with 5% CO2 for 2 h. The OD values of each well at a wavelength of 450 nm were measured using a microplate reader. The cytotoxicity of the compound was represented by cell viability, and the higher the cell viability, the lower the cytotoxicity. The results showed that the concentration (0 - 100 μM) had no cytotoxicity on mouse lung epithelial cells MLE-12. The experimental results are shown in Figure 5 .

[0074] Effect of Example 8 on the expression of cellular inflammatory factors IL-1β, IL-6, and TNF-α after LPS stimulation

[0075] Seeding plates and mRNA extraction

[0076] MLE-12 cells in good growth state were selected, gently blown down with cell culture medium, and the cell suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 3 min. After carefully aspirating the supernatant, the cells were resuspended, and cell counting was performed using a hemocytometer. 1×10 6 cells were seeded in each well of a 6-well plate and cultured in a carbon dioxide cell incubator for 24 h. Then, 5 groups were set, namely the blank control group (serum-free medium, Ctrl), the model group (serum-free medium containing 1 μg / mL LPS, Mod), the formononetin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L formononetin), the formononetin glucoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L formononetin glucoside), and the succinylformononetin glucoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L succinylformononetin glucoside).

[0077] The cells were pre-protected with the drug for 6 h, and then 1 μg / mL LPS was added for stimulation for 1 d. After discarding the cell culture medium, 1 mL of RNAiso Plus reagent was added, and the cells were repeatedly blown to fully lyse the cells. The cell lysate was transferred to an RNase-free 1.5 mL tube;

[0078] Then, chloroform (1 / 5 volume of RNAiso Plus) was added to the cell lysate, the lid of the EP tube was tightened, and the centrifuge tube was inverted up and down to fully mix until the solution became milky white, and then left standing for 15 min;

[0079] Centrifuge at 12,000 rpm at 4°C for 15 min. Carefully remove the EP tube from the low-temperature high-speed centrifuge. At this time, the homogenate is divided into three layers, namely, colorless supernatant (containing RNA), the middle white protein layer (mostly DNA), and the colored lower organic phase. Carefully aspirate the supernatant and transfer it to another new EP tube, taking care not to aspirate the white middle layer;

[0080] Precipitate RNA. Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube up and down to mix well, then let it stand at room temperature for 10 min. Centrifuge at 12,000 rpm at 4°C for 15 min. Observe that RNA precipitate appears at the bottom of the tube. Carefully discard the supernatant, taking care not to touch the precipitate;

[0081] Wash RNA. Add 500 μL of 75% ethanol, gently invert the tube up and down to wash the RNA precipitate, centrifuge at 9,000 rpm at 4°C for 5 min, discard the supernatant, taking care not to touch the precipitate, and repeat the RNA washing 2 times;

[0082] Dissolve RNA. Open the lid of the EP tube to dry the precipitate. Observe that the precipitate changes from white to transparent, and add an appropriate amount of DEPC-treated water to dissolve the precipitate;

[0083] RNA quantification and purity analysis. Take 2 μL of the dissolved RNA solution, use a multifunctional microplate reader to detect the concentration, and measure the absorbance values at wavelengths of 260 nm and 280 nm respectively. An OD260 / OD280 ratio between 1.8 - 2.1 indicates high RNA purity, OD260 / OD280 < 1.8 indicates possible residual protein impurities, and OD260 / OD280 > 2.1 indicates possible RNA degradation.

[0084] Reverse transcription

[0085] Reverse transcription reagent for mRNA IIQ RT SuperMix for qPCR (+gDNA wiper) was purchased from Nanjing Novoprotein Science and Technology Co., Ltd., product number R223, and is suitable for two-step qRT-PCR detection. The 4×gDNAwiper Mix in the product can completely remove the genomic DNA contamination remaining in the RNA template, and the procedure is 42°C for 2 min; the 5×HiScript qRT SuperMixⅡ contains all the components required for the reverse transcription reaction. Adding template RNA and water can initiate the reaction quickly, and at the same time terminate the action of gDNA wiper to ensure the integrity of cDNA. Reverse transcribe RNA into cDNA according to the kit instructions, and the reverse transcription system is 20 μL. The specific reverse transcription reaction system and reaction program are shown in Table 1.

[0086] Table 1 Reverse transcription reaction system and reaction program

[0087]

[0088] Note: The 4×gDNA wiper Mix and 5×HiScript qRT SuperMixⅡ contain a high concentration of glycerol. Before use, briefly centrifuge to collect at the bottom of the tube, and gently pipette to mix well before accurately aspirating.

[0089] Real-time fluorescence q-PCR

[0090] The Q-PCR reagent AceQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novoprotein Scientific Co., Ltd., with the product number Q511. It is a dedicated reagent for qPCR using the SYBR Green I chimeric fluorescence method. Prepare the q-PCR reaction system according to the kit instructions. The total system is 20 μL, and the specific volumes of each reactant are shown in Table 2.

[0091] Table 2 q-PCR reaction system

[0092]

[0093] Prepare the above reagents with each reactant according to the kit instructions. After thoroughly mixing by inverting up and down, centrifuge the reagents on the tube wall to the bottom of the tube, and place it in the StepOne Real Time PCR instrument. Perform the q-PCR reaction using the program shown in Table 3. For data analysis, normalize by subtracting the Ct value of β-actin in the same group from the Ct value of each gene, and perform conversion using the 2-△△Ct method to calculate the corresponding expression levels of each gene.

[0094] Table 3 q-PCR reaction conditions

[0095]

[0096] The primer sequences of each gene are shown in Table 4.

[0097] Table 4 q-PCR primer sequences

[0098]

[0099] The results showed that the expressions of IL-1β, IL-6, and TNF-α in the model group were significantly increased, showing significant differences compared with the normal control group (P<0.01). The expressions of IL-1β, IL-6, and TNF-α in the succinylformononetin group were significantly decreased, showing significant differences compared with the formononetin group (P<0.01). The experimental results are shown in Figure 6,The inhibitory effect of succinonaringenin on the expression of IL-1β, IL-6, and TNF-α in lung epithelial cells after LPS stimulation was increased to 250%-300% compared with that of formononetin, and the inhibitory effect of naringenin on the expression of IL-1β, IL-6, and TNF-α in lung epithelial cells after LPS stimulation was increased to 130%-200% compared with that of formononetin, which was statistically significant.

[0100] Effect of Example 9 on cell migration ability after LPS stimulation

[0101] Select MLE-12 cells with good growth status, gently blow the cells down with cell culture medium, collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, carefully aspirate the supernatant, resuspend the cells, count the cells with a hemocytometer, and seed 1×10 6 cells per well in a 6-well plate. After culturing in a carbon dioxide cell incubator for 24 h, set 5 groups, namely blank control group (serum-free medium, Ctrl), model group (serum-free medium containing 1 μg / mL LPS, Mod), formononetin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L formononetin), naringenin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L naringenin), and succinonaringenin group ((serum-free medium containing 1 μg / mL LPS and 25 μmol / L succinonaringenin). After the cells were completely confluent, use a sterile 10 μL pipette tip to perform cell scratching, wash twice with 1 mL of PBS to remove cell debris, then pre-protect the cells with the drug for 6 h, and then add 1 μg / mL LPS and continue to culture for 24 h. At the same time, set a blank control for each time period, and then use a cell imaging system to observe the cell migration at a magnification of 100 times. The results showed that the cell migration rate in the model group was significantly decreased, with a significant difference compared with the normal control group (P<0.01). The cell migration rate in the succinonaringenin group increased, with a significant difference compared with formononetin (P<0.05). The experimental results are shown in Figure 7 ,The promoting effect of succinonaringenin on the migration of lung epithelial cells after LPS stimulation was increased to 140% compared with that of formononetin, and the promoting effect of naringenin on the migration of lung epithelial cells after LPS stimulation was increased to 110% compared with that of formononetin, which was statistically significant.

[0102] Effect of Example 10 on cell apoptosis ability after LPS stimulation

[0103] Select MLE-12 cells with good growth status, gently blow the cells down with cell culture medium, collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, carefully aspirate the supernatant, resuspend the cells, count the cells with a hemocytometer, and seed 1×106 Cells were placed in a carbon dioxide incubator and cultured for 24 hours. Then, five groups were set up, namely the blank control group (serum-free medium, Ctrl), the model group (serum-free medium containing 1 μg / mL LPS, Mod), the formononetin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L formononetin), the ononin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L ononin), and the succinylononin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L succinylononin). When the cells grew to 75%-85% of the culture dish, the cells were pre-protected with the drug for 6 hours, then 1 μg / mL LPS was added, and the cells were cultured for another 24 hours. Trypsin was added to digest the cells (200 μL / well). After digestion, the cells were transferred to a 2 mL tube using a pipette gun, centrifuged at 5,000 rpm for 5 minutes, and the supernatant was discarded. 500 μL of PBS was added to each tube to resuspend the cells, and then centrifuged again to discard the supernatant. 195 μL of 1× binding buffer was accurately added to each tube, the cells were vortexed, and Annexin V-FITC staining solution (3 μL) and PI staining solution (2 μL) were accurately added. The cells were incubated in the dark at room temperature for 30 minutes. The cell suspension was slowly injected to the bottom of the flow tube, and immediately the apoptotic cells were detected by flow cytometry. Among them, Annexin V-FITC showed green fluorescence and PI showed red fluorescence. The results showed that the apoptosis rate of the model group was significantly decreased, and there was a significant difference compared with the normal control group (P<0.01). The apoptosis rate of the succinylononin group was significantly decreased, and there was a significant difference compared with the formononetin group (P<0.01). The experimental results are shown in Figure 8 , the inhibitory effect of succinylononin on the apoptosis of lung epithelial cells after LPS stimulation was increased to 120% compared with formononetin, and the promoting effect of ononin on the migration of lung epithelial cells after LPS stimulation was increased to 105% compared with formononetin, which was statistically significant.

[0104] Preparation of Tablets in Example 11

[0105] Prescription (for 1000 tablets):

[0106] 60 g of the pure succinylononin obtained in Example 2; 60 g of sucrose; 80 g of corn starch; 2 g of magnesium stearate.

[0107] Preparation method: The active ingredient was mixed with sucrose and corn starch, moistened with water, stirred evenly, dried, crushed and sieved, magnesium stearate was added, and mixed evenly, then pressed into tablets. The average tablet weight was 202 mg / tablet, and the content of the active ingredient was 60 mg.

[0108] Preparation of Injection in Example 6

[0109] Prescription (for a prescription volume of 1000 vials):

[0110] 10 g of the pure succinonaringenin obtained in Example 2; 100 g of propylene glycol; add water for injection to 1000 mL. Dissolve the prescription amount of pure succinonaringenin in propylene glycol, add water for injection to 1000 mL, mix evenly, filter, and aseptically dispense the obtained solution into ampoules to prepare an injection of 1 mL / vial with an active ingredient content of 10 mg / mL.

[0111] Matters not covered in this invention are well-known techniques.

[0112] The above examples are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. An application of a formononetin derivative, characterized in that: The use of the formononetin derivative in the preparation of a drug for treating and preventing acute lung injury, the structural formula of the formononetin derivative is as follows:

2. The use of the formononetin derivative according to claim 1, characterized in that: Acute lung injury includes hemoptysis, respiratory distress, respiratory failure, hypoxemia, and hypercapnia.

3. The use of the formononetin derivative according to claim 1, characterized in that: The formononetin derivative is the only active ingredient in the drug.

4. The use of the formononetin derivative according to claim 1, characterized in that: The medicine comprises a formononetin derivative and a pharmaceutically acceptable salt, ester, hydrate or combination thereof of the formononetin derivative and auxiliary materials.

5. The use of the formononetin derivative according to claim 1, characterized in that: The salt of the formononetin derivative refers to a salt formed by a formononetin derivative and hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, malonic acid, succinic acid, glutaric acid, maleic acid, alkyl or aryl sulfonic acid.

6. The use of the formononetin derivative according to claim 1, characterized in that: The effective therapeutic amount of succinylated formononetin is 10 mg / kg-40 mg / kg.

7. The use of the formononetin derivative according to claim 1, characterized in that: The dosage form of the drug is selected from tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, wine preparations, tinctures, dews, films or combinations thereof.

8. The use of the formononetin derivative according to claim 1, characterized in that: The administration methods of the drug include oral administration, injection, implantation, external application, spraying, inhalation or a combination thereof.

Images