Application of coix seed extract in preparation of medicine for preventing and treating acute lung injury

Abstract

The invention discloses an application of a coix seed extract in preparation of a medicine for preventing and treating acute lung injury. The semen coicis extract is obtained by performing reflux extraction on semen coicis by using ethanol, recovering an extraction solvent by using a rotary evaporator and then performing evaporation concentration by using a water bath. The coix seed extract can obviously increase the thymus index, improve the pathological injury of lung tissues, reduce the level of inflammatory factors in serum and lung tissues, possibly inhibit the expression of NLRP3, Caspase-1 and GSDMD-N in NLRP3 signal channel key proteins in the lung tissues, inhibit the NLRP3 signal channel, inhibit inflammation and pyroptosis, and play a role in protecting ALI. The coix seed extract can inhibit the activation of an NLRP3 signal channel and play a role in treating the acute lung injury disease.

Description

Technical Field

[0001] The present invention relates to a new application of a coix seed extract, and in particular to an application of the coix seed extract in preparing a medicine for preventing and treating acute lung injury. Background Art

[0002] Job's tears are the dried mature seed kernels of the grass plant Coix lacryma-jobi L.var.mayuen (Roman.)Stapf. In autumn, when the fruits are ripe, the plants are harvested, sun-dried, the fruits are beaten, and then sun-dried again. The shells, yellow-brown seed coats and impurities are removed, and the kernels are collected. It has the functions of promoting water and dampness, strengthening the spleen and relieving diarrhea, removing numbness, draining pus, and detoxifying and dispersing nodules. It is used for edema, beriberi, dysuria, spleen deficiency diarrhea, dampness and stiffness, lung abscess, intestinal abscess, warts, and cancer.

[0003] Acute lung injury (ALI) is an acute inflammatory response to the lungs caused by a variety of factors, typically manifesting as alveolar damage, impaired gas exchange, and hypoxemia. Its primary characteristic is an imbalance between ventilation and perfusion in the lungs, leading to oxygenation failure. ALI is the precursor to acute respiratory distress syndrome (ARDS), a more severe pathological condition. The pathogenesis of ALI is primarily related to two factors: one that leads to the accumulation of large amounts of protein and neutrophils in the pulmonary interstitium and alveoli, and the other that impairs the clearance of pulmonary edema fluid and inflammatory cells.

[0004] Although some progress has been made in the diagnosis, treatment and pathogenesis of ALI, due to its numerous pathogenic factors, complex pathogenesis and numerous links involved, there is still a lack of effective treatment measures. Therefore, ALI has always been a hot topic and difficulty in critical care medicine research at home and abroad. In the current existing technology, the clinical treatment of ALI is mainly based on mechanical ventilation and fluid management. To date, there is still a lack of effective therapeutic drugs for the multi-level pathogenesis of ALI, resulting in high morbidity and mortality. In summary, it is of great significance to find safe and effective therapeutic drugs that can prevent or treat acute lung injury caused by multiple factors.

[0005] Since the pharmacological substances of coix seed have not been fully elucidated, the purpose of the present invention is to study whether coix seed extract can be used to treat ALI. Research in this area has not been reported yet. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide the use of coix seed extract in the preparation of drugs for the prevention and treatment of acute lung injury. Experiments have proven for the first time that coix seed can significantly improve the general behavioral indicators of ALI mice, increase the thymus index, improve lung tissue pathological damage, and reduce the levels of inflammatory factors in serum and lung tissue. Coix seed extract may also inhibit the expression of key proteins NLRP3, Caspase-1 and GSDMD-N in the NLRP3 signaling pathway in lung tissue, inhibit the NLRP3 signaling pathway, inhibit inflammation and cell pyroptosis, and exert a protective effect against ALI. Coix seed extract has application prospects as a drug for the prevention and treatment of acute lung injury.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] Application of coix seed extract in the preparation of drugs for preventing and treating acute lung injury.

[0009] The preparation method of the coix seed extract specifically comprises the following steps:

[0010] (1) Take 2-3 kg of coix seed and extract it with 45% ethanol 1-3 times, soaking the coix seed in 13000-17000 mL of 45% ethanol for 10-30 minutes each time, heating it to boiling over high heat, then turning to low heat and keeping it slightly boiling for 0.5-2.0 hours. The color of the extract first turns light yellow and then fades to slightly milky white, then filter and combine the filtrates to obtain the coix seed extract;

[0011] (2) The coix seed extract is recovered and extracted using a rotary evaporator at a speed of 20-60 r / min and a temperature of 50-90° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 70-110° C. to obtain the coix seed extract.

[0012] In the above step (1), 2.3-2.7 kg of coix seed is taken and extracted with 45% ethanol 1-2 times, each time soaking the coix seed with 14000-16000 mL of 45% ethanol for 10-20 minutes, heating to boiling over high heat, then turning to low heat and maintaining a slight boil for 0.5-1.5 hours. The color of the extract first turns light yellow and then fades to slightly milky white, and then filtered, and the filtrates are combined to obtain a coix seed extract.

[0013] Specifically, in the aforementioned step (1), 2.5 kg of coix seed is taken and extracted with 45% ethanol 1-2 times, each time soaking the coix seed with 15000 mL of 45% ethanol for 15 minutes, heating to boiling over high heat, then turning to low heat and keeping it boiling for 1 hour, the color of the extract first turns light yellow and then fades to slightly milky white, then filtered, and the filtrates are combined to obtain the coix seed extract.

[0014] In the aforementioned step (2), the coix seed extract is recovered and extracted using a rotary evaporator at a speed of 25-55 r / min and a temperature of 60-80° C. to obtain a concentrated solution, and the concentrated solution is then evaporated to dryness of ethanol in a water bath at a temperature of 80-100° C. to obtain the coix seed extract.

[0015] Specifically, in the aforementioned step (2), the coix seed extract is recovered and extracted using a rotary evaporator at a speed of 30-50 r / min and a temperature of 65-75°C to obtain a concentrated solution, and the concentrated solution is then evaporated to dryness of ethanol in a water bath at a temperature of 85-95°C to obtain the coix seed extract.

[0016] The aforementioned acute lung injury includes acute lung injury caused by sepsis.

[0017] A medicine for preventing and treating acute lung injury, comprising coix seed extract.

[0018] A medicine for preventing and treating acute lung injury, wherein the active ingredient of the medicine comprises coix seed extract.

[0019] The preparation method of the aforementioned drug for preventing and treating acute lung injury is to combine coix seed extract with acceptable pharmaceutical excipients, and process them according to conventional preparation methods to prepare corresponding oral pharmaceutical preparations, which include tablets, capsules, pills, granules, powders and syrups.

[0020] The beneficial effects of the present invention are:

[0021] 1. This study used an LPS-induced ALI mouse model as the experimental subject. Experimental results demonstrated that coix seed extract can alleviate interstitial inflammatory cell infiltration in the lung tissue, increase the thymic index, and improve lung tissue pathological damage. It can significantly reduce the expression levels of the inflammatory factor TNF-α and IL-1β in lung tissue. Furthermore, it may inhibit the expression of NLRP3, Caspase-1, and GSDMD-N, key proteins in the NLRP3 signaling pathway, in lung tissue, thereby inhibiting the NLRP3 signaling pathway, suppressing inflammation and pyroptosis, and exerting a protective effect against ALI. This suggests that coix seed extract can treat acute lung injury and protect damaged lung tissue, and can be used as an active ingredient in drugs for the treatment of acute lung injury.

[0022] 2. After testing, the extract of coix seed was found to be rich in organic acids and amino acids. It can reduce the inflammatory factors TNF-α and IL-1β, inhibit NLRP3 inflammasome, reduce the expression levels of IL-18 and IL-1β in lung tissue, and inhibit inflammation and cell pyroptosis through the synergistic effect of the rich organic acids and amino acids.

[0023] 3. The present invention expands more targeted therapeutic drugs for the treatment of acute lung injury. The coix seed extract used can be used as a protective drug for acute lung injury. It has good efficacy, produces little toxic side effects, and has good application prospects.

[0024] 4. The present invention verifies through in vivo experiments that coix seed extract has a good protective effect on acute lung injury, greatly improves lung damage, inhibits inflammation and cell pyroptosis, and exerts a protective effect on ALI. This provides a theoretical basis for the treatment of ALI with coix seed extract and provides new ideas for the development of new ALI therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the body weight change of animals in each group (n=10);

[0026] Figure 2 The changes of organ index and lung dry-wet ratio of animals in each group (n=7-10);

[0027] Figure 3 Pathological sections of lung tissues of animals in each group (n=5);

[0028] Figure 4 Effects of coix seed extract on inflammatory factors in serum and lung tissue of ALI mice (n=9-10);

[0029] Figure 5 Effects of coix seed extract on NLRP3 signaling pathway in lung tissue of ALI mice (A is the representative protein band of each group; B is the expression of NLRP3 protein; C is the expression of Caspase-1 protein; D is the expression of GSDMD-N protein) (n=4);

[0030] Figure 6 This is the total ion current diagram of coix seed extract (A is the ion current diagram in positive ion mode, B is the ion current diagram in negative ion mode).

[0031] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention. DETAILED DESCRIPTION

[0032] Example 1:

[0033] Extraction process:

[0034] (1) Take 2.5 kg of coix seed and soak it in 15000 mL of 45% ethanol for 15 min. Heat it over high heat until it boils, then reduce the heat and keep it boiling for 1 h. The color of the extract first turns light yellow and then fades to slightly milky white. After filtering, add 45% ethanol and repeat the extraction twice. The filtrates are combined to obtain the coix seed extract.

[0035] (2) The coix seed extract was recovered and extracted using a rotary evaporator at a speed of 30-50 r / min and a temperature of 65-75° C. to obtain a concentrated solution, and the concentrated solution was evaporated to dry ethanol in a water bath at a temperature of 85-95° C. to obtain the coix seed extract.

[0036] Efficacy: Prevent or treat acute lung injury.

[0037] Usage: Take orally 0.4-0.8g daily.

[0038] Example 2:

[0039] Extraction process:

[0040] (1) 3 kg of coix seed was soaked in 13000 mL of 45% ethanol for 30 min, heated to boiling over high heat, then reduced to low heat and kept at a slight boil for 2 h. The color of the extract first turned light yellow and then faded to slightly milky white, then filtered, and then 45% ethanol was added for extraction and repeated three times. The filtrates were combined to obtain the coix seed extract;

[0041] (2) The coix seed extract is recovered and extracted using a rotary evaporator at a speed of 20-60 r / min and a temperature of 50-90° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 70-110° C. to obtain the coix seed extract.

[0042] Efficacy: Prevent or treat acute lung injury.

[0043] Usage: Take orally 0.4-0.8g daily.

[0044] Example 3:

[0045] Extraction process:

[0046] (1) 2 kg of coix seed was soaked in 17000 mL of 45% ethanol for 10 min, heated to boiling over high heat, then reduced to low heat and kept at a slight boil for 0.5 h. The color of the extract first turned light yellow and then faded to slightly milky white, then filtered, and the extraction was repeated with 45% ethanol once. The filtrates were combined to obtain the coix seed extract.

[0047] (2) The coix seed extract was recovered and extracted using a rotary evaporator at a speed of 30-50 r / min and a temperature of 65-75° C. to obtain a concentrated solution, and the concentrated solution was evaporated to dry ethanol in a water bath at a temperature of 85-95° C. to obtain the coix seed extract.

[0048] Efficacy: Prevent or treat acute lung injury.

[0049] Usage: Take orally 0.4-0.8g daily.

[0050] Example 4:

[0051] Extraction process:

[0052] (1) 2.2 kg of coix seed was soaked in 14000 mL of 45% ethanol for 20 min, heated to boiling over high heat, then reduced to low heat and kept at a slight boil for 1 h. The color of the extract first turned light yellow and then faded to slightly milky white, then filtered, and then 45% ethanol was added and extracted twice. The filtrates were combined to obtain the coix seed extract;

[0053] (2) The coix seed extract is recovered and extracted using a rotary evaporator at a speed of 25-55 r / min and a temperature of 60-80° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 80-100° C. to obtain the coix seed extract.

[0054] Efficacy: Prevent or treat acute lung injury.

[0055] Usage: Take orally 0.4-0.8g daily.

[0056] Example 5:

[0057] Extraction process:

[0058] (1) 2.7 kg of coix seed was soaked in 16000 mL of 45% ethanol for 15 min, heated to boiling over high heat, then reduced to low heat and kept at a slight boil for 1.5 h. The color of the extract first turned light yellow and then faded to slightly milky white, then filtered, and then extracted with 45% ethanol twice. The filtrates were combined to obtain the coix seed extract.

[0059] (2) The coix seed extract is recovered and extracted using a rotary evaporator at a speed of 20-60 r / min and a temperature of 50-90° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 70-110° C. to obtain the coix seed extract.

[0060] Efficacy: Prevent or treat acute lung injury.

[0061] Usage: Take orally 0.4-0.8g daily.

[0062] Example 6:

[0063] The coix seed extract obtained in Example 1 was used as a single active ingredient, and conventional pharmaceutical carriers or excipients were added to prepare tablets.

[0064] Efficacy: Prevent or treat acute lung injury.

[0065] Usage: Orally take 2-3 tablets per day. The content of coix seed extract is 0.2-0.3g per tablet.

[0066] Example 7:

[0067] The coix seed extract obtained in Example 1 was used as a single active ingredient, and conventional pharmaceutical carriers or excipients were added to prepare capsules.

[0068] Efficacy: Prevent or treat acute lung injury.

[0069] Usage: Orally take 2-3 tablets per day. The content of coix seed extract is 0.2-0.3g per tablet.

[0070] Example 8:

[0071] The coix seed extract obtained in Example 1 is used in combination with other medicinal ingredients for treating acute lung injury, and conventional pharmaceutical carriers or excipients are added to prepare pills.

[0072] Efficacy: Prevent or treat acute lung injury.

[0073] Dosage and administration: Orally take daily, 5-6 pills / time. The content of coix seed extract is 0.08-0.16g / pill.

[0074] Example 9:

[0075] The coix seed extract obtained in Example 1 is used in combination with other medicinal ingredients for treating acute lung injury, and conventional pharmaceutical carriers or excipients are added to prepare a powder.

[0076] Efficacy: Prevent or treat acute lung injury.

[0077] Dosage and administration: Oral administration daily, 5-6g / time. Each gram of powder contains 0.08-0.16g of coix seed extract.

[0078] Example 10:

[0079] The coix seed extract obtained in Example 1 is used in combination with other medicinal ingredients for treating acute lung injury, and conventional pharmaceutical carriers or excipients are added to prepare granules.

[0080] Efficacy: Prevent or treat acute lung injury.

[0081] Dosage and administration: Oral administration daily, 10g / time. Each gram of granules contains 0.04-0.08g of coix seed extract.

[0082] The inventors have conducted a large number of experiments, and the following is the research on the extraction method of the present invention:

[0083] 1. Drug efficacy experiment

[0084] 1.1 Materials and inventions

[0085] 1.1.1 Experimental animals

[0086] Sixty healthy male Kunming mice weighing 20-25 g were randomly selected. The experimental animals were provided by the Laboratory Animal Research Institute of Guizhou University of Traditional Chinese Medicine.

[0087] 1.1.2 Experimental drugs and instruments

[0088] Take 2.5 kg of coix seed, soak the coix seed in 15000 mL of 45% ethanol for 15 minutes, heat to boiling over high heat, then reduce heat and keep slightly boiling for 1 hour. The color of the extract first turns light yellow and then fades to slightly milky white, then filters, adds 45% ethanol and extracts repeatedly twice, and combines the filtrates to obtain coix seed extract; the coix seed extract is recovered and extracted using a rotary evaporator at a speed of 30-50 r / min and a temperature of 65-75°C to obtain a concentrated solution, and then the concentrated solution is evaporated to dryness of ethanol in a water bath at a temperature of 85-95°C to obtain a coix seed extract, which is stored at -20°C for later use.

[0089] Lipopolysaccharide (LPS) (Sigma-Aldrich Trading Co., Ltd., 0000263267), mouse TNF-α ELISA kit (Xinbosheng Biotechnology, M241015-102a), mouse IL-1β ELISA kit (Xinbosheng Biotechnology, M241015-001b), mouse IL-18 ELISA kit (Xinbosheng Biotechnology, M241015-011), dexamethasone (DEX, as a positive drug) (Huazhong Pharmaceutical Co., Ltd., H42041292).

[0090] A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-5210), a vacuum pump (SHB-895), an electronic balance (Hangzhou Wante Weighing Instrument Co., Ltd., WT20001), a vacuum drying oven (Tianjin Test Instrument Co., Ltd., DZ-2BC II), an intelligent constant temperature water bath (Changzhou Enpei Instrument Manufacturing Co., Ltd., HH-6S), a digital display electric heating mantle (Shanghai Lichen Bangxi Instrument Technology Co., Ltd., ZNHW 5000 mL), a metal bath (Scilogex, USA, HB120-S), a low-temperature centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd., Ministar), a multifunctional microplate reader (Thermo Scientific, USA, Varioska), and a water purifier (Suzhou Mai Ai Shi Electric Co., Ltd., IMS-25).

[0091] 1.1.3 Experimental Grouping, Modeling, and Processing

[0092] Sixty mice were randomly divided into a blank group, a model group, a dexamethasone-positive drug group (5 mg / kg), and coix seed extract low-, medium-, and high-dose groups (26, 52, and 104 mg / kg), with 10 animals in each group. Except for the dexamethasone-positive drug group, mice in the coix seed extract low-, medium-, and high-dose groups were gavaged with the drug (0.2 mL / 10 g). The blank and model groups received an equal volume of saline for 7 consecutive days. The dexamethasone-positive drug group received an intraperitoneal injection of 5 mg / kg dexamethasone on days 6 and 7. The animals were fasted for 12 hours. Thirty minutes after drug administration on day 7, mice in the coix seed extract low-, medium-, and high-dose groups and the model group were intraperitoneally injected with LPS (15 mg / kg, 0.1 mL / 10 g) to establish the model. The blank group received an equal volume of saline. Twelve hours after modeling, samples were collected and relevant parameters were measured.

[0093] 1.1.4 Observation of general condition and body weight of mice

[0094] Starting from the first day of administration, the general condition of the mice was observed and their body weight was measured daily. The changes in physical signs of the animals before and after LPS challenge were particularly observed on the seventh day of administration.

[0095] 1.1.5 Organ Index, Lung Dry-Wet Ratio, and Sampling of ALI Mice

[0096] Mice were bled by eye removal, and serum was separated and set aside. After the animals were sacrificed by cervical dislocation, the spleen, thymus, lungs, heart, liver, and kidneys were removed and weighed, and organ indices were calculated. A portion of the right lung was weighed and oven-dried at 68°C for 48 hours. Lung relative water content = [lung wet weight (g) - lung dry weight (g)] / lung wet weight (g) × 100%. A portion of the lung tissue was fixed with 4% neutral formaldehyde, and the remaining lung tissue was cooled with liquid nitrogen and stored at -80°C until further use.

[0097] 1.1.6 Preparation of HE pathological sections of ALI mouse lung tissue

[0098] Lung tissues were fixed in 4% paraformaldehyde for 48 h, and then subjected to routine procedures such as ethanol dehydration, xylene clearing, paraffin embedding, sectioning, and hematoxylin-eosin staining. Colon HE pathological sections were prepared and observed under a microscope, and images were retained.

[0099] 1.1.7 Determination of inflammatory factors in serum and lung tissue

[0100] Serum was collected and the levels of inflammatory factors TNF-α and IL-1β were determined by ELISA according to the instructions of the assay kit. Lung tissue was collected, homogenized and centrifuged to obtain the supernatant, and the expression levels of IL-1β and IL-18 in the lung tissue were determined according to the steps of the ELISA kit.

[0101] 1.1.8 Western blot analysis of the expression of key proteins in the NLRP3 signaling pathway in lung tissue

[0102] A small amount of lung tissue from each group of animals was collected, ground into a powder using liquid nitrogen, and then homogenized with lysis buffer. The tissue was lysed on ice and centrifuged to separate the protein supernatant for protein concentration determination. After adjusting the protein samples to equal concentrations, the samples were loaded, electrophoresed, transferred to a membrane, blocked, incubated with primary and secondary antibodies, developed, and exposed to light. The expression of NLRP3, Caspase-1, and GSDMD-N proteins in the lungs was determined. The grayscale value of the target protein band divided by the grayscale value of the internal reference protein band was calculated as the relative protein content.

[0103] 1.1.9 Data Analysis

[0104] Data are expressed as mean ± standard error The data were expressed in the form of , and SPSS Statistics 25.0 software was used for statistical analysis. If the data showed a normal distribution, one-way ANOVA was used, and post hoc multiple comparisons were performed. If the homogeneity of variance was assumed, "LSD" was selected, and if the homogeneity of variance was not assumed, "Dunnett T3" was selected. If the data did not show a normal distribution, nonparametric tests were used for analysis.

[0105] 1.2 Results

[0106] 1.2.1 General behavioral observations and weight changes of mice

[0107] Before modeling, mice in all groups had smooth fur, were relatively active, responsive, and had regular breathing. After modeling, all groups except the blank group showed signs of sluggish reactions, disheveled fur, and rapid breathing. Compared with the model group, mice in the low-, medium-, and high-dose coix seed extract groups performed better overall and recovered better. The medium-dose coix seed extract group showed the fastest recovery of activity and was more responsive.

[0108] Before LPS attack, the weight of animals in each group increased slowly, and the weight of animals in the dexamethasone-positive drug group increased more slowly. After LPS attack, the weight of animals in the dexamethasone-positive drug group decreased significantly (see Figure 1 ).

[0109] 1.2.2 Organ index and lung dry-wet ratio of mice

[0110] Compared with the blank group, the liver and kidney indexes of the model group animals increased significantly (P<0.01). 26mg / kg and 104mg / kg coix seed extract had the effect of increasing the thymus index of ALI mice (P<0.05), and 52mg / kg coix seed extract had a tendency to increase the thymus index (see Figure 2 Coix seed extract had no significant effect on the heart, liver, spleen, lung, kidney indexes and lung dry-wet ratio of ALI mice.

[0111] 1.2.3 Observation of drug effects on lung tissue based on HE pathological sections

[0112] The pleural structure of the lung tissue of mice in the blank group was relatively normal, with thin walls and no connective tissue hyperplasia or thickening; the bronchial structures at all levels were complete and clear, the epithelial cell morphology was normal, and there was no obvious degeneration, necrosis or shedding; the type I and type II alveolar epithelial cells in the respiratory part of the lung were relatively normal in morphology, without obvious degeneration or necrosis, and no obvious inflammatory cell infiltration and fibrosis were observed in the interstitium. The lung tissue of animals in the model group showed vascular congestion, hyperplasia of type II alveolar epithelial cells and infiltration of interstitial inflammatory cells. The pathological changes in the lung tissue of animals in the dexamethasone-positive drug group and the low-, medium- and high-dose coix seed extract groups were alleviated to varying degrees. Among them, the interstitial inflammatory cell infiltration of the lung tissue of the high-dose coix seed extract group was alleviated, and no type II alveolar epithelial cell hyperplasia was observed in the medium-dose coix seed extract group (see Figure 3 ).

[0113] 1.2.4 Effects of coix seed extract on inflammatory factors in serum and lung tissue of ALI mice

[0114] Compared with the blank group, the inflammatory factors TNF-α and IL-1β in the serum and IL-18 and IL-1β in the lung tissue of the model group were significantly increased (P<0.01). After intervention with coix seed extract, it was found that coix seed extract (26, 52, 104 mg / kg) could significantly reduce the expression levels of TNF-α in serum and IL-1β in lung tissue (P<0.01). In addition, 104 mg / kg coix seed extract could significantly reduce the expression of IL-1β in serum (P<0.05), and coix seed extract (52, 104 mg / kg) could significantly reduce the expression level of IL-18 in lung tissue (P<0.01) (see Figure 4).

[0115] 1.2.5 Effect of coix seed extract on the expression of key proteins in the NLRP3 signaling pathway in the lungs of ALI mice

[0116] Compared with the blank group, the expression of NLRP3, Caspase-1 and GSDMD-N, key proteins of the NLRP3 signaling pathway, in the lungs of ALI mice in the model group were significantly increased, indicating that the NLRP3 signaling pathway in the lung tissue was activated. After intervention with coix seed extract, it was found that coix seed extract (52, 104 mg / kg) could significantly reduce the expression of NLRP3, Caspase-1 and GSDMD-N proteins in lung tissue (P<0.01). 26 mg / kg coix seed extract had a downward trend in the expression of NLRP3, Caspase-1 and GSDMD-N proteins in lung tissue, but there was no statistical significance. The above results indicate that coix seed extract can inhibit the activation of the NLRP3 signaling pathway in the lungs of ALI mice and inhibit the inflammatory response in the lungs of ALI mice (see Figure 5 ).

[0117] 2 Identification of Coix Seed Extract by LC-MS

[0118] 2.1 Instruments

[0119] Thermo Scientific Q Exactive Orbitrap LC-MS / MS (Thermo Fisher Scientific, USA); Waters ACQUITYUPLC HSS T3 C 18 Chromatographic column (2.1 mm × 100 mm, 1.8 μm).

[0120] 2.2 Detection method

[0121] 10 mg of coix seed extract was accurately weighed and dissolved in a 5 mL Eppendorf tube. An appropriate amount of methanol was added and sonicated. The resulting solution was centrifuged at 15,000 rpm for 20 minutes and then filtered through a 0.22 μm microporous membrane to obtain the test solution. The components of the coix seed extract were then identified using a Thermo Scientific Q Exactive Orbitrap LC-MS / MS (Thermo Fisher Scientific, USA). Chromatographic conditions: The mobile phase consisted of 0.05% acetic acid in water (A) and acetonitrile (B) at a flow rate of 0.3 mL / min, and the injection volume was 2 μL. The elution gradient was as follows: 5% to 30% B (0-20 min); 30% to 40% B (20-27 min); 40% to 70% B (27-32 min); 70% to 95% B (32-42 min); and 95% to 95% B (42-45 min). The column temperature was 30°C, and the detection wavelength (UV) was 254 nm. Mass spectrometry conditions included: full mass resolution = 35,000, dd-MS2 resolution = 17,500, scan range = 100-1500 m / z, spray voltage = 3.5 kV (positive) or 3.0 kV (negative). Ion transfer tube temperature was 350°C, auxiliary gas heating temperature was 300°C, sheath gas flow rate was 40 arb, and auxiliary gas flow rate was 15 arb. Stepped NCE was 20, 40, and 60 eV.

[0122] Compound Discover 3.0 software was used to match the molecular formula and relative molecular mass of the experimental data with the mzCloud and mzVault databases, with a primary and secondary mass deviation of 5×10 -6 , the matching score is >80, and the components are inferred and identified by combining the literature and fragment ion information.

[0123] 2.3 Results:

[0124] After the coix seed extract was tested, the total ion current of positive and negative ion scanning was obtained (see Figure 6 ), a total of 80 compounds were identified by liquid chromatography-mass spectrometry analysis, among which organic acids were relatively abundant, such as D-α-hydroxyglutaric acid, 1,2,3-cyclopropanetricarboxylic acid, 4-pyridoxic acid, 4-acetylaminobutyric acid, pantothenic acid, gentisic acid, adipic acid, trans-3-indoleacrylic acid, 2,3,4,9-tetrahydro-1H-β-carbene-3-carboxylic acid, and pimelic acid.

[0125] Table 1 Components in coix seed extract

[0126]

[0127]

[0128]

Claims

1. Application of coix seed extract in the preparation of drugs for the prevention and treatment of acute lung injury.

2. The use of the coix seed extract according to claim 1 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: The preparation method of the coix seed extract specifically comprises the following steps: (1) Take 2-3 kg of coix seed and extract it with 45% ethanol 1-3 times, soaking the coix seed in 13000-17000 mL of 45% ethanol for 10-30 minutes each time, heating it to boiling over high heat, then turning to low heat and keeping it slightly boiling for 0.5-2.0 hours. The color of the extract first turns light yellow and then fades to slightly milky white, then filter and combine the filtrates to obtain the coix seed extract; (2) The coix seed extract is recovered and extracted using a rotary evaporator at a speed of 20-60 r / min and a temperature of 50-90° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 70-110° C. to obtain the coix seed extract.

3. The use of the coix seed extract according to claim 2 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: In the step (1), 2.3-2.7 kg of coix seed is taken and extracted with 45% ethanol 1-2 times, each time soaking the coix seed with 14000-16000 mL of 45% ethanol for 10-20 minutes, heating to boiling over high heat, then turning to low heat and maintaining a slight boil for 0.5-1.5 hours, the color of the extract first turns light yellow and then fades to slightly milky white, and then filtered, and the filtrates are combined to obtain a coix seed extract.

4. The use of the coix seed extract according to claims 2 and 3 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: In the step (1), 2.5 kg of coix seed is extracted with 45% ethanol 1-2 times, each time immersing the coix seed in 15000 mL of 45% ethanol for 15 minutes, heating to boiling over high heat, then turning to low heat and keeping it slightly boiling for 1 hour, the color of the extract first turns light yellow and then fades to slightly milky white, and then filtered, and the filtrates are combined to obtain a coix seed extract.

5. The use of the coix seed extract as claimed in claim 2 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: In the step (2), the coix seed extract is recovered and extracted using a rotary evaporator at a speed of 25-55 r / min and a temperature of 60-80° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 80-100° C. to obtain the coix seed extract.

6. The use of the coix seed extract according to claim 2 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: In the step (2), the coix seed extract is recovered and extracted using a rotary evaporator at a speed of 30-50 r / min and a temperature of 65-75° C. to obtain a concentrated solution, and the concentrated solution is evaporated to dry ethanol in a water bath at a temperature of 85-95° C. to obtain the coix seed extract.

7. The use of the coix seed extract according to claim 1 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: The acute lung injury includes acute lung injury caused by sepsis.

8. A drug for preventing and treating acute lung injury, characterized by: The medicine includes coix seed extract.

9. A drug for preventing and treating acute lung injury, characterized by: The active ingredient of the medicine includes coix seed extract.

10. The method for preparing the drug for preventing and treating acute lung injury according to claims 8 and 9, characterized in that: The preparation method comprises combining coix seed extract with acceptable pharmaceutical excipients, and processing the mixture according to a conventional preparation method to prepare corresponding oral pharmaceutical preparations, which include tablets, capsules, pills, granules, powders and syrups.

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