Use of formononetin derivative in preparation of drug for preventing and treating acute lung injury

Abstract

Disclosed is use of formononetin-7-O-(6-O-succinyl)-D-glucoside in the preparation of a drug for treating and preventing acute lung injury, which can reduce the occurrence and progression of lung injury in animal models, and can attenuate inflammatory damage to lung epithelial cells in vitro.

Description

Application of a gentianin derivative in the preparation of drugs for the prevention and treatment of acute lung injury Technical Field

[0001] This invention relates to a novel use of a mangiferin derivative in medicine, specifically to the use of a mangiferin derivative in the preparation of drugs for the treatment and prevention of acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is an acute, diffuse inflammatory disease of the lungs caused by various etiologies. It manifests as damage to the alveolar-capillary membrane, pulmonary edema, impaired gas exchange, and refractory hypoxemia, leading to a rapid decline in lung function and potentially progressing to acute respiratory distress syndrome (ARDS). ALI has extremely high morbidity and mortality rates, posing a significant threat to public health. Current interventions for ALI primarily involve protective mechanical ventilation, anti-inflammatory drugs, and corticosteroids such as dexamethasone, pulmonary vasodilators, and statins. While these treatments can be effective, they also present a range of side effects to varying degrees.

[0003] Flavonoids are products of plant secondary metabolism, diverse in type and structure, exhibiting rich pharmacological activities and playing an important role in the prevention and treatment of cardiovascular and respiratory diseases. Mangosteen, as one of the representative flavonoids, displays a variety of pharmacological activities, regulating respiratory inflammation and preventing respiratory diseases by inhibiting airway inflammation. However, mangosteen has low water solubility, which limits its use to some extent. A mangosteen derivative—succinomangosteen glycoside—has higher water solubility and is less toxic and safer. Furthermore, succinomangosteen glycoside can effectively treat cardiovascular diseases, but its application in respiratory diseases has not yet been studied. The key to this invention is comparing the efficacy of the mangosteen derivative—succinomangosteen glycoside—in preventing and treating acute lung injury compared to mangosteen itself. Summary of the Invention

[0004] The purpose of this invention is to provide a new use of a mangiferin derivative in medicine, specifically the application of a mangiferin derivative in the preparation of drugs for the treatment and prevention of acute lung injury.

[0005] The objective of this invention is achieved through the following technical solution: The existing compound succinyl monzoline is monzoline-7-O-(6-O-succinyl)-D-glucoside, and the structure of the obtained succinyl monzoline is shown in Formula 1:

[0006] Formula 1:

[0007] This invention provides the application of a gentianin derivative in the preparation of drugs for the treatment and prevention of acute lung injury.

[0008] Preferably, acute lung injury includes hemoptysis, respiratory distress, respiratory failure, hypoxemia, and hypercapnia.

[0009] Preferably, the gentianin derivative in the drug is the sole active ingredient.

[0010] Preferably, the drug comprises aromonin derivatives and pharmaceutically acceptable salts, esters, hydrates, or combinations thereof of aromonin derivatives, as well as excipients.

[0011] Preferably, the mangiferin derivative salt refers to a salt formed by mangiferin derivative with 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.

[0012] Preferably, the effective therapeutic dose of succinyl mangiferin is 10 mg / kg to 40 mg / kg.

[0013] Preferably, the dosage form of the drug is selected from tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, tinctures, lotions, films, or combinations thereof.

[0014] Preferably, the drug is administered orally, by injection, implantation, external application, spray, inhalation, or a combination thereof. Beneficial effects

[0015] 1. The succinyl mangiferin of the present invention inhibits lung inflammation and lung pathological changes, and reduces the levels of related cytokines in lung tissue and bronchoalveolar lavage fluid.

[0016] 2. The succinyl mangiferin of the present invention protects against inflammatory damage to lung epithelial cells, promotes migration, and inhibits apoptosis.

[0017] 3. The experimental results of the water solubility analysis of gentianin, gentianin derivatives—gentianoside, and succinylgentianoside in this invention show that:

[0018] Succinyl mangiferin exhibits significantly improved water solubility compared to mangiferin. The results show that succinyl mangiferin's water solubility is approximately 822 times higher than that of mangiferin.

[0019] 4. The results of mouse experiments in this invention show that:

[0020] (1) The effect of succinyl mangiferin derivatives on improving the degree of lung injury in mice in the LPS-induced acute lung injury model was increased by 200% compared with mangiferin, and the effect of mangiferin on improving the degree of lung injury in mice in the LPS-induced acute lung injury model was increased by 130% compared with mangiferin, which was statistically significant.

[0021] (2) The effect of succinyl mangosteen derivatives on improving lung pathological changes and reducing lung pathological scores in model mice was increased by 250% compared with mangosteen, and the effect of mangosteen glycoside on improving lung pathological changes and reducing lung pathological scores in model mice was increased by 130% compared with mangosteen, which is statistically significant.

[0022] (3) The derivative of mangosteen, succinyl mangosteen, reduced the effects of mangosteen on cytokines IL-4, IL-5, and IL-13 in mouse bronchoalveolar lavage fluid by 200%-250%, and mangosteen glycoside reduced the effects of mangosteen on cytokines IL-4, IL-5, and IL-13 in mouse bronchoalveolar lavage fluid by 130%-180%, which is statistically significant.

[0023] 5. This invention demonstrates through in vitro lung epithelial cell culture experiments that:

[0024] (1) Succinyl mangiferin derivative—succinyl mangiferin has no toxic effect on lung epithelial cells.

[0025] (2) The derivative of mangosteen—succinyl mangosteen glycoside—increased the inhibition of IL-1β, IL-6, and TNF-α expression in LPS-stimulated lung epithelial cells by 250%-300% compared with mangosteen, and the inhibition of IL-1β, IL-6, and TNF-α expression in LPS-stimulated lung epithelial cells by 130%-200% compared with mangosteen, which was statistically significant.

[0026] (3) The derivative of mangosteen—succinodyl mangosteen glycoside—increased the effect of mangosteen on promoting the migration of lung epithelial cells after LPS stimulation by 140%, and the effect of mangosteen glycoside on promoting the migration of lung epithelial cells after LPS stimulation by 110%, which is statistically significant.

[0027] (4) The derivative of mangosteen—succinyl mangosteen glycoside—inhibited lung epithelial cell apoptosis by 120% compared with mangosteen, and the effect of mangosteen glycoside on promoting lung epithelial cell migration after LPS stimulation by 105% compared with mangosteen, which was statistically significant. Attached Figure Description

[0028] Figure 1 shows the content of gentianin in licorice residue;

[0029] Figure 2 shows the comparison of lung dry weight / wet weight in mice of each experimental group;

[0030] Figure 3 shows a comparison of lung pathology in mice from different experimental groups (X200);

[0031] Figure 4 shows the changes in the levels of IL-4, IL-5, and IL-13 in the airway lavage fluid of mice in each experimental group;

[0032] Figure 5 shows the effect of different doses of mangiferin derivatives on cell proliferation.

[0033] Figure 6 shows the effects of gentianin, gentianin, and succinylgentianin on the expression of inflammatory cytokines IL-1β, IL-6, and TNF-α after LPS stimulation.

[0034] Figure 7 shows the effects of gentianin, gentianin, and succinylgentianin on cell migration after LPS stimulation (X100).

[0035] Figure 8 shows the effects of gentianin, gentianin, and succinylgentianin on apoptosis after LPS stimulation. Detailed Implementation

[0036] To further understand the present invention, preferred embodiments of the present invention are 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, and not for limiting the scope of the claims of the present invention.

[0037] Example 1: Efficiently obtaining gentianin compounds from licorice residue

[0038] Extracting flavonoids from Chinese medicinal herb residue includes the following steps:

[0039] Place 5g of licorice residue in a 250mL Erlenmeyer flask, add 3g / L (NH4)2SO4, 0.5g / L urea, 2g / L KH2PO4, 0.5g / L MgSO4, 0.1% metal ions (FeSO4·7H2O, 0.005g / L, MnSO4·H2O, 0.0016g / L, ZnSO4·7H2O, 0.0014g / L, CoCl2, 0.002g / L), and 100mL pure water. Sterilize in a 121℃ autoclave for 20 minutes. After cooling to 28℃, inoculate with *Penicillium oxalicum* Z2 (CCTCC NO: M 20241501) preserved at the China Center for Type Culture Collection (CCTCC) (inoculation amount 1wt%, post-inoculation concentration 1×10⁻⁶). 6The licorice residue extract was prepared by fermentation (CFU / mL) under the following conditions: temperature 28℃, rotation speed 200 rpm, fermentation time 4 days. After fermentation, the temperature was raised to 55℃ for in-situ enzymatic hydrolysis for 10 hours, followed by incubation at 90℃ for 15 minutes. After hydrolysis, the residue was cooled to room temperature and centrifuged at 5000 rpm. The extract was then filtered through 200-mesh gauze to obtain the licorice residue extract.

[0040] Preparative liquid chromatography (HPLC) was performed using an XB-C18 micrometer (5 μm particle size, 250 mm length, 10 mm inner diameter) at a flow rate of 3 mL / min. A mobile phase of methanol and formic acid-water was used, with isocratic elution at a mass ratio of 60:40. The retention time of gentianin was approximately 14.56–15.12 min, and its content was approximately 30% of the licorice residue extract. The experimental results are shown in Figure 1. Subsequently, the fraction was concentrated to a small volume using a rotary evaporator at 40 °C, and then freeze-dried to obtain a solid powder compound. The purity of the components was determined by HPLC-UV to ensure that the purity was above 90%.

[0041] The compounds isolated from the extract were utilized 1 The structure was determined using methods such as H-NMR and in conjunction with references. The obtained structure of stylosanol is shown in Equation 2.

[0042] Formula 2:

[0043] Manggenin: a white amorphous powder. 1 H-NMR(400MHz,DMSO-d6)δppm 3.79(S,3H)6.87(d,J=2.08Hz,1H)6.94(dd,J=8.80,2.20Hz,1H)6.99(m,8.80Hz,2H)7.97(d,J=8.68Hz,1H)8.34(s,1H)10.80(br.s.,1H)

[0044] In this embodiment, a total of 0.29 grams of gentianin solid powder was obtained from 5 grams of licorice residue.

[0045] Example 2: Obtaining monganin and succinylmonganin via biotransformation

[0046] The fermentation culture of *Bacillus amyloliquefaciens* FJ18 was inoculated into a seed culture medium containing 5.0 g / L yeast extract, 10.0 g / L peptone, 10.0 g / L NaCl, and pH 7.0, and cultured at 30°C and 200 rpm for 12 hours. The expansion and fermentation media contained the following components and concentrations: 20 g / L sucrose, 15 g / L yeast extract, 1.0 g / L KH₂PO₄, and 0.8 g / L CaCl₂. The pH was adjusted to 8.0 with NaOH. The seed culture was inoculated into the expansion and fermentation media at 0.5% (v / v) and cultured at 30°C and 200 rpm for 12 hours. After centrifugation at 10,000 rpm for 15 minutes, the bacterial cells were collected and washed 1-2 times with physiological saline to obtain resting *Bacillus amyloliquefaciens* FJ18 cells.

[0047] The fermentation broth was filtered to obtain wet bacterial cells. A reaction solution was prepared using dimethyl sulfoxide (DMSO), gentianin, sucrose, and phosphate buffer. The reaction solution contained 20% (v / v) DMSO, 0.5 g / L gentianin, 150 mmol / L phosphate buffer, pH 8.0, and 50 g / L sucrose. The obtained wet bacterial cells were dispersed in the reaction solution and added to a reactor. After incubation at 30°C and 200 rpm for 24 h, the supernatant was obtained by centrifugation at 10,000 rpm for 10 min. HPLC analysis showed a gentianin conversion rate of 97.2%.

[0048] The product was separated using macroporous resin. An appropriate amount of resin was soaked in ethanol for 24 hours, and resin fragments and impurities were removed. The wet-packed column was rinsed with 1 L of ethanol, then washed with distilled water until no alcohol odor remained. Acid-base treatment was then performed: the resin column was passed sequentially with 5% (v / v) HCl solution and 2% (w / w) NaOH solution at a flow rate of 2 BV / h, and allowed to stand for 2–4 hours. The column was then washed with distilled water until the pH was neutral. To avoid dissolving the conversion product in DMSO and reducing the adsorption rate, the conversion solution was diluted with 5 times its volume of deionized water (pH 4.0, adjusted with glacial acetic acid) to DMSO < 2% before loading. The loading volume was 20 mg / g wet resin at a flow rate of 20 mL / min. Excess unreacted glycosyl donors (sucrose) were rinsed with 10 times the column bed volume of deionized water (pH 4.0, adjusted with glacial acetic acid) until the eluent was undetectable by concentrated sulfuric acid at a flow rate of 20 mL / min. Methanol and deionized water were used as the mobile phase for elution. The volume ratio of methanol to deionized water was adjusted, and the proportion of methanol in the eluent was determined at an elution flow rate of 20 mL / min. Concentration and drying: The eluents were combined and concentrated under reduced pressure using a rotary evaporator at 40 °C. Finally, the solid was placed in a vacuum drying oven and dried at 40 °C for 6 h.

[0049] The chemical formula for the preparation of succinyl monigrin from Bacillus amyloliquefaciens FJ18 in a non-aqueous phase is shown below:

[0050] Nuclear magnetic resonance mass spectrometry (NMR) analysis confirmed that the structure obtained was consistent with the expected product. These results confirm that the product generated in this reaction...

[0051] The glucosylated mangiferin, namely succinyl mangiferin. The NMR spectral data of succinyl mangiferin are as follows:

[0052] 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).

[0053] 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”’).

[0054] The structure of the obtained product, gentianin, is shown in Formula 3:

[0055] Formula 3:

[0056] The structure of the obtained product, succinyl monzoline, is shown in Formula 1:

[0057] Example 3: Water solubility analysis of monganin, monganin, and succinylmonganin

[0058] A standard curve was established using HPLC, and the solubility of saturated aqueous solutions of gentianin, gentianoside, and succinoside was determined at 37 °C. Results: The water solubility of gentianin was 0.017 g / L, that of gentianoside was 6.106 g / L, and that of succinoside was 13.967 g / L. These results indicate that the water solubility of succinoside was approximately 822 times higher than that of gentianin.

[0059] Example 4: Effect on the improvement of lung injury in mice

[0060] 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) Mangiferin (F) group; (5) Mangiferin (FG) group; (6) Succinyl mangiferin (FMP) group. The mice in the treatment groups were administered dexamethasone (5 mg / kg), mangiferin group (40 mg / kg), mangiferin group (40 mg / kg), and succinyl mangiferin group (40 mg / kg) by gavage, respectively. The blank control group and the model group were administered the corresponding volume of physiological saline by gavage. The administration was carried out continuously for 3 days. One hour after the last administration, 10 mg / kg LPS was dripped into the nasal cavity to establish an acute lung injury model in mice. Twenty-four hours after the last challenge, the animals were sacrificed, and the middle and lower lobes of the right lungs of the mice were harvested. Surface moisture was thoroughly absorbed with filter paper, and the mass was recorded as wet weight using an analytical balance. The tissues were then dried in a 60℃ constant-temperature drying oven until constant weight, and the mass of the lung tissue was recorded as dry weight. The W / D ratio was calculated using the following formula. The experimental results are shown in Figure 2. The degree of lung injury in the model group was significantly increased compared to the normal control group (P<0.01). The degree of lung injury in the succinyl mangiferin group was reduced, increasing by 200% compared to mangiferin, which was statistically significant (P>0.01, P<0.05). The effect of mangiferin in improving the degree of lung injury in the LPS-induced acute lung injury model in mice was increased by 130% compared to mangiferin.

[0061] Example 5: Improvement effect on lung pathological changes and reduction of lung pathological scores in mice.

[0062] 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) Mangiferin (F) group; (5) Mangiferin (FG) group; (6) Succinyl mangiferin (FMP) group. The mice in the treatment groups were administered dexamethasone (5 mg / kg), mangiferin (40 mg / kg), mangiferin (40 mg / kg), and succinyl mangiferin (40 mg / kg) by gavage, respectively. The blank control group and the model group were administered the corresponding volume of physiological saline by gavage. The administration was repeated for 3 days. One hour after the last administration, 10 mg / kg LPS was instilled into the nasal cavity to establish an acute lung injury model in mice. Twenty-four hours after the last challenge, the animals were sacrificed, and their lungs were harvested and fixed in 10% formalin. Paraffin-embedded sections were prepared, approximately 4–5 μm thick, and stained with hematoxylin and eosin (HE). Based on the severity of the lesions, they are semi-quantitatively classified as mild ("+"), moderate ("++"), severe ("++++"), and no lesion tissue ("-").

[0063] In the model group, the thickness of bronchial smooth muscle was significantly increased (+++), with moderate exudate in the lumen; a large number of inflammatory cells (+++) were infiltrated in the submucosa and muscular layer of the trachea, mainly including eosinophils, lymphocytes, macrophages, and a small number of plasma cells. Partial shedding of the bronchial ciliated epithelium was observed, with cilia adhering, collapsing, degenerating, and necrosis; multilayered airway epithelium (+++). In the dexamethasone group, the thickness of bronchial smooth muscle was slightly increased (+), with no exudate in the lumen; a small number of eosinophils, lymphocytes, and a small number of plasma cells were infiltrated in the submucosa and muscular layer of the trachea (+). The pulmonary lobule structure was generally normal, with mild adhesion, collapsing, and degeneration of the bronchial cilia (+). In the gentianin group, the thickness of bronchial smooth muscle was moderately increased (++), with moderate exudate in the lumen; inflammatory cell infiltration (++) was observed in the submucosa and muscular layer of the trachea, mainly including eosinophils, lymphocytes, macrophages, and a small number of plasma cells. Partial shedding of the bronchial ciliated epithelium, with cilia adhering, collapsing, and degenerating, and multilayering of the airway epithelium (++) were observed. In the gentianin group, the thickness of bronchial smooth muscle was slightly increased (+), with exudate in the lumen; inflammatory cell infiltration (+) was observed in the submucosa and muscular layer of the trachea, mainly including eosinophils, lymphocytes, macrophages, and a small number of plasma cells. Partial shedding of the bronchial ciliated epithelium, with cilia adhering, collapsing, degenerating, and necrosis (++), and multilayering of the airway epithelium (++) were observed. In the succinyl mangosteen glycoside group, the thickness of bronchial smooth muscle was slightly increased (+), and exudate was present in the lumen. Inflammatory cell infiltration (+) was observed in the submucosa and muscular layer of the trachea, mainly including eosinophils, lymphocytes, macrophages, and a small number of plasma cells. Partial shedding of bronchial ciliated epithelium, ciliary adhesion, collapse, degeneration, and necrosis (+), and multilayered airway epithelium (+) were observed. The comparison of lung pathology in mice of each experimental group is shown in Figure 3. The effect of succinyl mangosteen glycoside on improving lung pathological changes and reducing lung pathological scores in model mice was increased by 250% compared with mangosteen glycoside, and the effect of mangosteen glycoside on improving lung pathological changes and reducing lung pathological scores in model mice was increased by 130% compared with mangosteen glycoside, which was statistically significant.

[0064] Example 6: Improvement effect on the expression of inflammatory factors IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid

[0065] 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) Mangiferin (F) group; (5) Mangiferin (FG) group; (6) Succinyl mangiferin (FMP) group. The mice in the treatment groups were administered dexamethasone (5 mg / kg), mangiferin group (40 mg / kg), mangiferin group (40 mg / kg), and succinyl mangiferin group (40 mg / kg) by gavage, respectively. The blank control group and the model group were administered the corresponding volume of physiological saline by gavage. The administration was carried out continuously for 3 days. One hour after the last administration, 10 mg / kg LPS was dripped into the nasal cavity to establish an acute lung injury model in mice. Twenty-four hours after the last challenge, the animals were euthanized, the thoracic cavity was opened, the trachea was exposed, and the right bronchus was ligated with cotton thread. Then, 0.8 mL of pre-cooled phosphate-buffered saline (PBS) was injected into the left lung via the main bronchus using a 1 mL syringe, and this was repeated three times. The lavage fluid was collected and stored at -80℃ for later use. The levels of IL-4, IL-5, and IL-13 cytokines were detected by ELISA. The results showed that the expression of IL-4, IL-5, and IL-13 in the model group was significantly increased compared with the normal control group (P<0.01). The succinyl mangiferin group showed decreased IL-4 expression, which was statistically significant compared with the mangiferin group (P>0.01, P<0.05), significantly decreased IL-5 expression, which was significantly different compared with the mangiferin group (P<0.01), and decreased IL-13 expression, which was significantly different compared with the mangiferin group (P<0.05). The experimental results are shown in Figure 4. Succinyl mangiferin reduced the effects of mangiferin on cytokines IL-4, IL-5, and IL-13 in mouse bronchoalveolar lavage fluid by 200%-250% compared to mangiferin, and mangiferin reduced the effects of mangiferin on cytokines IL-4, IL-5, and IL-13 in mouse bronchoalveolar lavage fluid by 130%-180%, which was statistically significant.

[0066] Example 7: Toxicity of lung epithelial cells

[0067] Mouse lung epithelial cells (MLE-12) were selected for cytotoxicity testing. Cells were digested with trypsin and counted, and the cell concentration was adjusted to 1 × 10⁻⁶. 5MLE-12 was seeded at concentrations of 0-10 μmol / mL in 96-well plates of DMEM / F12 high-glucose medium containing 10% fetal bovine serum and then cultured in a 37°C, 5% CO2 incubator for 24 h. Eight concentration groups were set up: 0, 1.5, 3, 6.25, 12.5, 25, 50, and 100 μmol / L, with three replicates per group. 24 h after drug administration, 10 μL of CCK-8 solution was added to each well, followed by 2 h of further culture in a 37°C, 5% CO2 incubator. The OD value of each well was measured at 450 nm using a microplate reader. The cytotoxicity of the compound was expressed as cell viability; higher cell viability indicated lower cytotoxicity. The results showed that concentrations (0-100 μM) had no cytotoxic effect on mouse lung epithelial cells (MLE-12). The experimental results are shown in Figure 5.

[0068] Example 8: Effects of LPS-stimulated cell inflammatory cytokines IL-1β, IL-6, and TNF-α expression

[0069] Seeding plate and mRNA extraction

[0070] Select MLE-12 cells in good growth condition, gently blow the cells off 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, and count the cells using a hemocytometer. Seed 1×10⁶ cells per well in a 6-well plate. 6 Cells were cultured in a CO2 cell culture incubator for 24 hours and then divided into five groups: blank control group (serum-free medium, Ctrl), model group (serum-free medium containing 1 μg / mL LPS, Mod), mangiferin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L mangiferin), mangiferin glycoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L mangiferin), and succinyl mangiferin glycoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L succinyl mangiferin).

[0071] After pre-protecting the cells with the drug for 6 hours, the cells were stimulated with 1 μg / mL LPS for 1 day. The cell culture medium was then discarded, and 1 mL of RNAiso Plus reagent was added. The cells were repeatedly pipetted to fully lyse them. The cell lysate was then transferred to an RNase-free 1.5 mL tube.

[0072] Next, add chloroform (1 / 5 volume of RNAiso Plus) to the cell lysis buffer, tighten the cap of the EP tube, invert the centrifuge tube, mix thoroughly until the solution turns milky white, and let stand for 15 minutes.

[0073] Centrifuge at 12000 rpm at 4℃ for 15 min. Carefully remove the EP tube from the low-temperature high-speed centrifuge. At this point, the homogenate will be divided into three layers: a colorless supernatant (containing RNA), a white protein layer in the middle (mostly DNA), and a colored lower organic phase. Carefully aspirate the supernatant and transfer it to another new EP tube, being careful not to aspirate the white middle layer.

[0074] To precipitate RNA, add an equal volume of isopropanol to the supernatant, invert the centrifuge tube to mix thoroughly, let stand at room temperature for 10 minutes, centrifuge at 12000 rpm at 4℃ for 15 minutes, observe the RNA precipitate at the bottom of the tube, carefully discard the supernatant, and do not touch the precipitate;

[0075] Wash RNA. Add 500 μL of 75% ethanol, gently invert the container to wash the RNA precipitate, centrifuge at 9000 rpm at 4℃ for 5 min, discard the supernatant, and do not touch the precipitate. Repeat the RNA washing process twice.

[0076] Dissolve the RNA. Open the EP tube cap and allow the precipitate to dry. Observe that the precipitate changes from white to transparent. Add an appropriate amount of DEPC-treated water to dissolve the precipitate.

[0077] RNA quantification and purity analysis: 2 μL of the dissolved RNA solution was taken, and the concentration was detected using a multi-functional microplate reader. The absorbance was measured at wavelengths of 260 nm and 280 nm, respectively. An OD260 / OD280 ratio between 1.8 and 2.1 indicated high RNA purity; OD260 / OD280 < 1.8 indicated possible residual protein impurities; and OD260 / OD280 > 2.1 indicated possible RNA degradation.

[0078] Reverse transcription

[0079] mRNA reverse transcription reagent The IIQ RT SuperMix for qPCR (+gDNA wiper) was purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number R223, and is suitable for two-step qRT-PCR detection. The 4×gDNA wiper Mix in the product thoroughly removes residual genomic DNA contamination from the RNA template; the program is 42℃ for 2 min. The 5×HiScript qRT SuperMix II contains all the components required for the reverse transcription reaction; adding template RNA and water initiates a rapid reaction, simultaneously terminating the gDNA wiper action to ensure cDNA integrity. RNA was reverse transcribed into cDNA according to the kit instructions, with a reverse transcription volume of 20 μL. The specific reverse transcription reaction system and program are shown in Table 1.

[0080] Table 1. Reverse transcription reaction system and reaction procedure

[0081] Note: 4×gDNA wiper Mix and 5×HiScript qRT SuperMixⅡ contain a high concentration of glycerol. Before use, they should be briefly centrifuged to collect the contents to the bottom of the tube, and then gently pipetted to mix thoroughly before accurately aspirating.

[0082] Real-time fluorescence q-PCR

[0083] The AceQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd. (catalog number Q511). It is a dedicated reagent for qPCR using the SYBR Green I chimeric fluorescence assay. The qPCR reaction system was prepared according to the kit instructions, with a total volume of 20 μL. The specific volumes of each reactant are shown in Table 2.

[0084] Table 2 q-PCR reaction system

[0085] Prepare the reagents according to the kit instructions, thoroughly mix by inverting the tube, centrifuge to remove the reagents from the tube wall, and place the tube in a StepOne Real-Time PCR instrument. Perform q-PCR using the program shown in Table 3. Data analysis: Normalize by subtracting the corresponding Ct value of each gene from the Ct value of β-actin in the same group, convert using the 2-ΔΔCt method, and then calculate the expression level of each gene.

[0086] Table 3 q-PCR reaction conditions

[0087] The primer sequences for each gene are shown in Table 4.

[0088] Table 4 q-PCR primer sequences

[0089] The results showed that the expression of IL-1β, IL-6, and TNF-α in the model group was significantly increased compared with the normal control group (P<0.01). The expression of IL-1β, IL-6, and TNF-α in the succinyl mangosteenin group was significantly decreased compared with the mangosteenin group (P<0.01). The experimental results are shown in Figure 6. Succinyl mangosteenin significantly increased the inhibition of IL-1β, IL-6, and TNF-α expression in LPS-stimulated lung epithelial cells by 250%-300% compared with mangosteenin, and mangosteenin significantly increased the inhibition of IL-1β, IL-6, and TNF-α expression in LPS-stimulated lung epithelial cells by 130%-200% compared with mangosteenin, which was statistically significant.

[0090] Example 9: Effect of LPS-stimulated cell migration ability

[0091] Select MLE-12 cells in good growth condition, gently blow the cells off 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, and count the cells using a hemocytometer. Seed 1×10⁶ cells per well in a 6-well plate. 6 Cells were cultured in a CO2 cell culture incubator for 24 hours and then divided into five groups: a blank control group (serum-free medium, Ctrl), a model group (serum-free medium containing 1 μg / mL LPS, Mod), a gentianin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L gentianin), a gentianoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L gentianoside), and a succinyl gentianoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L succinyl gentianoside). After complete cell confluence, cell scratching was performed using a sterile 10 μL pipette tip, followed by washing twice with 1 mL PBS to remove cell debris. Cells were then pre-protected with medication for 6 hours before adding 1 μg / mL gentianoside. LPS was used to culture the cells for another 24 hours, with blank controls performed at each time point. Cell migration was then observed using a cell imaging system at 100x magnification. The results showed that the cell migration rate in the model group was significantly reduced compared to the normal control group (P<0.01). The cell migration rate in the succinyl mangiferin group was significantly increased compared to mangiferin (P<0.05). The experimental results are shown in Figure 7. Succinyl mangiferin increased the migration rate of LPS-stimulated lung epithelial cells by 140% compared to mangiferin, and mangiferin increased the migration rate of LPS-stimulated lung epithelial cells by 110% compared to mangiferin, which was statistically significant.

[0092] Example 10: Effect of LPS-stimulated cells on apoptosis capacity

[0093] Select MLE-12 cells in good growth condition, gently blow the cells off 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, and count the cells using a hemocytometer. Seed 1×10⁶ cells per well in a 6-well plate. 6Cells were cultured in a CO2 cell culture incubator for 24 hours. Five groups were then established: a blank control group (serum-free medium, Ctrl), a model group (serum-free medium containing 1 μg / mL LPS, Mod), a gentianin group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L gentianin), a gentianoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L gentianoside), and a succinyl gentianoside group (serum-free medium containing 1 μg / mL LPS and 25 μmol / L succinyl gentianoside). When the cells reached 75%-85% confluence in the culture dish, they were pre-protected with the drug for 6 hours before adding 1 μg / mL LPS. 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 2 mL tubes using a pipette. The cells were centrifuged at 5,000 rpm for 5 min, and the supernatant was discarded. 500 μL of PBS was added to each tube to resuspend the cells. The cells were centrifuged again, and the supernatant was discarded. 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 at room temperature in the dark for 30 min. The cell suspension was slowly injected into the bottom of a flow cytometry tube, and the number of apoptotic cells was immediately detected using a flow cytometer. Annexin V-FITC staining solution was added to each well. V-FITC showed green fluorescence, and PI showed red fluorescence. The results indicated that the apoptosis rate in the model group was significantly reduced compared to the normal control group (P<0.01). The apoptosis rate in the succinyl mangiferin group was also significantly reduced compared to mangiferin (P<0.01). The experimental results are shown in Figure 8. Succinyl mangiferin significantly increased the inhibition of LPS-stimulated lung epithelial cell apoptosis by 120% compared to mangiferin, and significantly increased the promotion of LPS-stimulated lung epithelial cell migration by 105% compared to mangiferin, which were statistically significant.

[0094] Example 11 Preparation of tablets

[0095] Prescription (based on a prescription of 1000 tablets):

[0096] The following ingredients were used in Example 2: 60g of pure succinyl monazine; 60g of sucrose; 80g of corn starch; and 2g of magnesium stearate.

[0097] Preparation method: Mix the active ingredient with sucrose and corn starch, moisten with water, stir evenly, dry, pulverize and sieve, add magnesium stearate, mix evenly, and compress into tablets. The average tablet weight is 202 mg / tablet, and the active ingredient content is 60 mg.

[0098] Example 6: Preparation of Injection Solution

[0099] Prescription (based on a prescription of 1000 vials):

[0100] 10g of the pure succinyl monazine obtained in Example 2; 100g of propylene glycol; and water for injection to a final volume of 1000mL. The prescribed amount of pure succinyl monazine was dissolved in propylene glycol, and water for injection was added to a final volume of 1000mL. The mixture was thoroughly mixed, filtered, and the resulting solution was aseptically dispensed into ampoules to prepare a 1mL / ampoule injection solution with an active ingredient content of 10mg / mL.

[0101] Matters not covered in this invention are common knowledge.

[0102] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of a gentianin derivative, characterized in that: The application of the gentianin derivative in the preparation of drugs for treating and preventing acute lung injury, wherein the structural formula of the gentianin derivative is as follows:

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

3. The application of the gentianin derivative according to claim 1, characterized in that: The drug contains a gentianin derivative as its sole active ingredient.

4. The application of the gentianin derivative according to claim 1, characterized in that: The drug comprises a mangiferin derivative and pharmaceutically acceptable salts, esters, hydrates, or combinations thereof of mangiferin derivatives, as well as excipients.

5. The application of the gentianin derivative according to claim 1, characterized in that: The sophoroside derivative salt refers to the salt formed by sophoroside derivatives with 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 application of the gentianin derivative according to claim 1, characterized in that: The effective therapeutic dose of succinyl mangiferin is 10 mg / kg-40 mg / kg.

7. The application of the gentianin 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, tinctures, lotions, films, or combinations thereof.

8. The application of the gentianin derivative according to claim 1, characterized in that: The drug can be administered orally, by injection, implantation, external application, spray, inhalation, or a combination thereof.

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