Application of composition in preparation of medicine for preventing or treating acute lung injury

Abstract

The invention discloses application of a composition in preparation of a medicine for preventing or treating acute lung injury, and relates to the field of natural medicines. The pneumonia mixture can significantly improve the general state, weight change, lung coefficient, lung W / D ratio, pathological change, blood routine and biochemical indexes of rats with the LPS-induced acute lung injury, and shows that the pneumonia mixture has a certain protective effect on the rats with the lipopolysaccharide-induced acute lung injury. In addition, the pneumonia mixture has good safety. Therefore, the pneumonia mixture can be used for preventing and treating acute lung injury, has good curative effect and small toxic and side effects, and has good prospect value.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and in particular to application of a composition in preparing a medicine for preventing or treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a common respiratory critical illness in clinical practice. Its occurrence and development involve multiple pathophysiological processes. It is characterized by rapid onset and fierce onset, mainly manifested by progressively worsening hypoxic respiratory failure. It is a type of acute respiratory distress syndrome (ARDS) with milder symptoms. If not treated promptly and effectively, it can develop into ARDS or even death. The morbidity and mortality of ALI / ARDS are very high. It has been reported that the incidence of ALI / ARDS in the population is 57-78 cases per 100,000 people per year, and the mortality rate is 35% to 46%. It seriously threatens the life of patients and affects their quality of life, bringing a heavy burden to the family and society.

[0003] Uncontrolled inflammatory response is the intrinsic cause of the occurrence and progression of ALI. The secretion of proinflammatory mediators increases, and ALI can cause abnormal damage to pulmonary capillary endothelial cells and alveolar epithelial cells. Its characteristic pathological changes are pulmonary edema, hyaline membrane formation, and pulmonary fibrosis caused by increased pulmonary microvascular permeability. The essence of this change is the loss of control and imbalance after significant and continuous cascade expansion of the inflammatory response in the lungs, which in turn leads to damage to the pulmonary vascular endothelial barrier, alveolar epithelial barrier, and acute respiratory dysfunction.

[0004] Inflammation plays an important role in the occurrence and development of ALI / ARDS. Therapeutic methods that inhibit the inflammatory cascade can provide a promising strategy for the prevention and treatment of ALI / ARDS. Common inflammation comes from Gram-negative bacterial infection. The outer membrane is the main pathogenic factor of the cell wall of Gram-negative bacteria. Lipopolysaccharide (LPS) is the main component of endotoxins in the outer membrane. It can induce increased secretion of pro-inflammatory mediators, causing significant and continuous cascade expansion of inflammatory reactions in the lungs, leading to severe inflammatory reactions. LPS is a key factor in the pathophysiology of ALI. The animal or cell models induced by LPS have similar pathological damage degrees and time courses as ALI, and can simulate ALI to a large extent. It can be used to screen candidate drugs for the prevention and treatment of ALI.

[0005] At present, the treatment of ALI includes primary disease treatment, respiratory support therapy, drug therapy, etc., and some progress has been made. Among them, mechanical ventilation therapy is the main treatment method and is relatively effective, but it is easy to cause secondary damage to lung tissue; existing therapeutic drugs include glucocorticoids, nitric oxide (NO) inhalation, alveolar surfactant, prostaglandin E1, etc., but different degrees of side effects or drug resistance make it difficult to identify specific drugs for the treatment of ALI / ARDS. Due to the complex pathogenesis, the treatment effect of existing methods is not ideal, and it is obviously not enough for the prevention and treatment of ALI / ARDS. Therefore, it is urgent to find a treatment method with significant efficacy, few side effects, and conducive to improving prognosis.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a composition for use in preparing a drug for preventing or treating acute lung injury to solve the above technical problems.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides an application of a composition in the preparation of a drug for preventing or treating acute lung injury, the composition comprising the following raw materials in weight percentage: 5-20% scutellaria baicalensis, 1-15% licorice, 1-10% ephedra, 10-25% isatis root, 10-20% gypsum, 5-15% bitter almond, 15-25% reed root and 10-20% honeysuckle; the preparation method of the composition comprises: extracting the raw materials with water, concentrating the filtrate obtained by filtration, then precipitating the concentrate with alcohol, and collecting the ethanol liquid obtained by the alcohol precipitation.

[0010] The present invention has the following beneficial effects:

[0011] The present invention finds that the prescription used by clinicians for treating pneumonia in children has the effect of preventing or treating acute lung injury. By studying the rat model of acute lung injury induced by lipopolysaccharide (LPS), it is found that the Chinese medicine composition can significantly improve the general state of rats with acute lung injury induced by LPS, improve weight loss, improve the increase of lung coefficient, improve the increase of lung W / D ratio, improve the pathological changes of lung tissue, and improve abnormal blood routine and abnormal biochemical indicators.

[0012] By conducting acute toxicity tests on the composition, it was found that the composition had no obvious acute toxicity, was safe and reliable for oral administration, had a wide safety range, and had good clinical application safety.

[0013] Therefore, the composition can be used for the prevention and treatment of acute lung injury, has good efficacy, small toxic and side effects, and has good prospect value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 It is the total ion current chromatogram of the pneumonia mixture of the present invention (the abscissa is the retention time, and the ordinate is the electrical signal on the detector);

[0016] Figure 2 The effect of the pneumonia mixture of the present invention on the weight change ratio of ALI rats;

[0017] Figure 3 The effect of the pneumonia mixture of the present invention on the lung coefficient of ALI rats;

[0018] Figure 4 The effect of the pneumonia mixture of the present invention on the W / D ratio of the lung tissue of ALI rats;

[0019] Figure 5 The effect of the pneumonia mixture of the present invention on the gross observation and pathological changes (HE staining) of the lung tissue of ALI rats;

[0020] Figure 6 The effect of the pneumonia mixture of the present invention on the red blood cell parameters of ALI rats;

[0021] Figure 7 The effect of the pneumonia mixture of the present invention on the leukocyte classification count of ALI rats;

[0022] Figure 8 The effect of the pneumonia mixture of the present invention on platelet parameters in ALI rats;

[0023] Fig. 9 The effect of the pneumonia mixture of the present invention on the biochemical indexes of ALI rats;

[0024] Fig.10 The pneumonia mixture of the present invention affects the morphology of organs in healthy ICR mice;

[0025] Fig.11 The effect of the pneumonia mixture of the present invention on the body weight change curve of healthy ICR mice;

[0026] Fig.12 The pneumonia mixture of the present invention affects the organ coefficients of healthy ICR mice;

[0027] Fig.13 The invention discloses the effects of the pneumonia mixture on the blood biochemical indexes of healthy ICR mice. DETAILED DESCRIPTION

[0028] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0029] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0030] The term "acute lung injury" (ALI) refers to "acute lung injury", "disease" or "symptom". It is a common clinical respiratory critical illness. Its occurrence and development involve multiple pathophysiological processes. It is characterized by rapid onset and fierce onset, mainly manifested by progressively worsening hypoxic respiratory failure. It is a type of acute respiratory distress syndrome (ARDS) with milder symptoms.

[0031] The terms "disease" or "disorder" refer to disorders and / or abnormalities that are generally regarded as pathological conditions or functions and that may manifest themselves in the form of specific signs, symptoms, and / or malfunctions.

[0032] The term "treating" a disease or condition means eliminating, inhibiting, alleviating or relieving the disease or condition, and the term "preventing" means avoiding and preventing the disease or condition or causing the disease or condition not to occur or appear.

[0033] The term "pneumonia mixture" is synonymous with "Chinese medicine composition" or "composition".

[0034] In a first aspect, the present invention provides an application of a composition in the preparation of a drug for preventing or treating acute lung injury, the composition comprising the following raw materials in weight percentage: 5-20% scutellaria baicalensis, 1-15% licorice, 1-10% ephedra, 10-25% isatis root, 10-20% gypsum, 5-15% bitter almond, 15-25% reed root and 10-20% honeysuckle; the preparation method of the composition comprises: extracting the raw materials with water, concentrating the filtrate obtained by filtration, then precipitating the concentrate with alcohol, and collecting the ethanol liquid obtained by the alcohol precipitation.

[0035] Experiments have shown that the development of inflammation in the lungs of rats after LPS induction has a certain regularity, and after treatment, the inflammation is alleviated. The positive drug dexamethasone has an ameliorative effect on the lung injury of the ALI rat model induced by LPS, and the pre-administration and therapeutic administration of the Chinese medicine composition provided by the present invention can also play a protective role on rats with lung injury by directly regulating mechanisms such as inflammation, effectively controlling the development of inflammation, reducing the level of inflammation, reducing lung oxidative damage, and improving the expression of related inflammatory factors, playing a positive role in relieving lung inflammation, and providing a reference for the prevention or treatment of acute lung injury.

[0036] In addition, the composition provided by the present invention has no obvious acute toxicity, is safe and reliable for oral administration, has a wide safety range, and has good clinical application safety.

[0037] In a preferred embodiment of the present invention, the above-mentioned drug has at least one of the following uses:

[0038] (1) Improve the weight loss of subjects;

[0039] (2) Improvement of organ damage in subjects;

[0040] (3) improving the subject's pulmonary edema and / or congestion state;

[0041] (4) reduce the wet / dry weight ratio of the subjects' lungs;

[0042] (5) restore the color of the subject's lung tissue;

[0043] (6) Reduce the bleeding points and necrotic lesions in the subjects' lung tissue;

[0044] (7) reducing capillary congestion in the subject's lung tissue;

[0045] (8) improving alveolar septal thickening in the subject's lung tissue;

[0046] (9) improving inflammatory cell infiltration in the lung tissue of the subject;

[0047] (10) Improve the abnormal blood routine test results of the subjects;

[0048] (11) Improve the abnormal biochemical indicators of the subjects.

[0049] Abnormal blood test or abnormal biochemical index is relative to normal subjects. Abnormal blood test includes but is not limited to significant changes in the content of peripheral blood leukocytes, lymphocytes and hemoglobin, lymphocyte percentage and neutrophil percentage.

[0050] Abnormalities in blood biochemistry include but are not limited to abnormal changes in serum aspartate aminotransferase, triglycerides, creatine kinase, lactate dehydrogenase levels, TNF-α, IL-1β, protein, malondialdehyde (MDA) content, and myeloperoxidase (MPO) content or activity.

[0051] In other embodiments, the above-mentioned drug has the following uses: significantly improving the general condition (hair gloss, mental state, breathing rate, activity, diet, weight change) of subjects with LPS-induced acute lung injury, such as alleviating symptoms.

[0052] In a preferred embodiment of the present invention, the above-mentioned improvement of the abnormal blood routine test results of the subject includes at least one of the following methods:

[0053] (1) Improve the subject's red blood cell abnormalities;

[0054] (2) improving the subjects’ leukocyte abnormalities;

[0055] (3) Improve platelet abnormalities in subjects.

[0056] In a preferred embodiment of the present invention, the above-mentioned improvement of the abnormal blood routine test results of the subject includes at least one of the following methods:

[0057] (1) Reduce hemoglobin (HGB) level, hematocrit (HCT), neutrophil count (Neu) or ratio, intermediate cell count (Mon) or ratio, and platelet distribution width (PDW);

[0058] (2) Increased white blood cell (WBC) count or ratio, lymphocyte (Lym) count or ratio, platelet (PLT) count or ratio, and platelet count (PCT).

[0059] In a preferred embodiment of the present invention, the above-mentioned improvement of abnormal blood routine test results of the subject includes the following methods:

[0060] Reduce the levels of TNF-α, IL-1β, protein, malondialdehyde (MDA) and myeloperoxidase (MPO) activity in bronchoalveolar lavage fluid.

[0061] In a preferred embodiment of the present invention, the subject is a human or a non-human mammal, including but not limited to mice, rats, monkeys, and sheep.

[0062] In a preferred embodiment of the present invention, the acute lung injury is induced by LPS.

[0063] In a preferred embodiment of the present invention, the above-mentioned medicine further comprises a pharmaceutically acceptable carrier.

[0064] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from one or more of starch, pregelatinized starch, microcrystalline cellulose, lactose, lactose starch complex, lactose cellulose complex, mannitol, mannitol starch complex, sorbitol, povidone, copovidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, magnesium stearate, sodium stearyl fumarate, talc, and stearic acid.

[0065] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as solution, suspension and gel usually contain liquid carrier, such as water and / or pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include pH adjusting agents, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (such as methylcellulose), such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be regulated by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be regulated by pharmaceutically acceptable thickeners such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

[0066] In a preferred embodiment of the present invention, the dosage form of the above-mentioned drug is selected from any one of an oral preparation and an injection.

[0067] Compositions include but are not limited to: solid, liquid or semi-solid.

[0068] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0069] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0070] The experimental materials involved in the present invention are as follows:

[0071] Materials and Reagents:

[0072] Dexamethasone sodium phosphate injection (Henan Runhong Pharmaceutical Co., Ltd.); lipopolysaccharide and malondialdehyde (MDA) content detection kits were purchased from Beijing Solebold Technology Co., Ltd.; rat TNF-α Elisa kit, BCA protein concentration determination kit, and myeloperoxidase (MPO) colorimetric test kit were purchased from Wuhan Elaruite Biotechnology Co., Ltd.; IL-1β Elisa kit (Boster Bioengineering Co., Ltd.); alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (Cr) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute; methanol and acetonitrile were both LC-MS grade and purchased from CNW Technologies; ammonium acetate (LC-MS grade, SIGMA-ALDRICH); ammonia water (LC-MS grade, Fisher Chemical); ultrapure water (ddH2O) (Watsons).

[0073] Experimental animals: SD rats, weighing 220-330 g; ICR mice, weighing (25±5) g, half male and half female, all provided by the Animal Experiment Center of Xinjiang Medical University, license number [SCXK(Xin)2018-0002].

[0074] Main instruments and equipment:

[0075] Centrifuge (Sorvall ST 16R, Thermo Fisher Scientific); microscope (Eclipse Ni-U, Nikon); veterinary automatic blood cell analyzer (BC-5000Vet, Mindray); ELISA detector (MULTISKAN GO, Thermo Scientific); balance (BS224S, Sartorius); refrigerator (DW-HL678S, Meiling); water bath (B2kw, Beijing Yongxingming Medical Instrument Factory); vacuum drying oven (DZF-6090, Shanghai Yiheng); centrifuge (Sorvall ST 16R, Thermo Fisher Scientific); ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher); high-resolution mass spectrometer (QExactive HFX, Thermo Fisher); centrifuge (Heraeus Fresco17, Thermo Fisher Scientific); Scientific); balance (BSA124S-CW, Sartorius); ultrasonic instrument (PS-60AL, Shenzhen Redbond Electronics Co., Ltd.).

[0076] Example 1

[0077] This embodiment provides a method for preparing a Chinese medicine composition (pneumonia mixture), which specifically comprises the following steps:

[0078] The pneumonia mixture is provided by the preparation room of the First Affiliated Hospital of Xinjiang Medical University. It is prepared from eight traditional Chinese medicines including scutellaria baicalensis, licorice, ephedra, isatis root, gypsum, bitter almond, reed root, and honeysuckle through water extraction, filtration, concentration, alcohol precipitation, ethanol recovery, and addition of preservatives.

[0079] The prescription of pneumonia mixture includes: 550g of gypsum, 600g of reed root, 450g of bitter almond, 100g of ephedra, 650g of isatis root, 350g of honeysuckle, 150g of licorice, and 400g of scutellaria.

[0080] [Preparation method] Add 3 times the amount of water to the above eight ingredients, extract twice, 2 hours each time, filter, combine the filtrates, concentrate to 800ml at normal pressure, let it cool to room temperature, add 1000ml of 95% ethanol, let it stand at room temperature for 48 hours in summer and 24 hours in winter, recover the ethanol until the liquid has no alcohol taste; add 12ml of 2% paraben and 8ml of 10% benzoic acid ethanol, stir well, add water to 1000ml, and divide into packages.

[0081] Example 2

[0082] In this example, the chemical components of the prepared pneumonia mixture were analyzed based on UHPLC-QE-MS technology.

[0083] 100 μL of pneumonia mixture was transferred to an EP tube, and 400 μL of extract (methanol:acetonitrile = 1:1 (V / V), containing an isotope-labeled internal standard mixture) was added, vortexed for 30 s, ultrasonicated for 10 min (ice-water bath), allowed to stand for 1 h (-40 °C), and centrifuged for 15 min (4 °C, 12000 rpm); the supernatant was taken into an injection bottle for detection. Chromatographic conditions: ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm, Waters) was used, with an aqueous solution (A) containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia and acetonitrile (B) as the mobile phase, gradient elution (see Table 1), sample plate temperature: 4 °C, injection volume: 2 μL. Mass spectrometry conditions: Thermo QExactive HFX mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of control software (Xcalibur, Thermo). The detailed parameters are as follows: sheath gas flow rate: 30Arb, auxiliary gas flow rate: 25Arb, capillary temperature: 350°C, full MS resolution: 120000Da, MS / MS resolution: 7500Da, collision energy in NCE mode: 10 / 30 / 60ev, spray voltage: 3600V(+), 3200V(-).

[0084] Table 1 Elution gradient

[0085]

[0086] The total ion current chromatogram obtained is as follows Figure 1 As shown. The chemical components of pneumonia mixture were qualitatively identified by combining the self-built secondary mass spectrometry database of BiotreeDB (V2.1) and consulting literature and reference material information for comparison. The retention time, molecular formula, molecular ion peak, and fragment information results were listed in the order of retention time. A total of 123 chemical components were identified in the positive and negative ion modes. Some of the results are shown in Table 2.

[0087] Table 2 Identification of the main components of pneumonia mixture

[0088]

[0089]

[0090]

[0091]

[0092] Note: *Compounds confirmed by reference substances

[0093] Example 3

[0094] This example conducts a pharmacodynamic study of the pneumonia mixture based on an LPS-induced acute lung injury model in rats.

[0095] 1. Animal Grouping

[0096] SD rats were randomly divided into 5 groups, with 6 rats in each group: blank control group, model group, positive drug dexamethasone group, pneumonia mixture pre-administration group, and pneumonia mixture treatment group.

[0097] 2. Preparation of Rat ALI Model

[0098] The rats were weighed and recorded for calculation of the dose of anesthetic drugs and lipopolysaccharide. Rats were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.3 g / kg). The rats were anesthetized and their breathing slowed down and their limb muscles relaxed. They were fixed on the operating table with adhesive tape and the front teeth of the rats were fixed with rubber bands to make the skin in front of the neck tight. The skin was disinfected with 75% alcohol 3 times. The skin was longitudinally incised along the midline of the front neck (the incision was about 1.0 cm long). The muscle layer in front of the trachea was carefully bluntly separated to expose the trachea. The trachea was fixed with forceps in the left hand, and the indwelling needle was held in the right hand parallel to the direction of the trachea and slowly inserted into the trachea toward the heart. After confirming that the tracheal wall had been broken through, the needle core was withdrawn, and the needle tip was withdrawn to the soft cannula. The soft cannula was slightly pushed forward horizontally against the trachea, and LPS (15 mg / ml, 15 mg / kg) or an equal volume of saline was slowly dripped into the trachea. After the intratracheal instillation is completed, withdraw the needle core, push in 300 μL of air to reduce stagnation in the tube, quickly stand the rat up, and rotate it rapidly with the body as the central axis so that the drug can be distributed as evenly as possible in the two lungs through the centrifugal force. Keep the rat in the upright position for 1 min. Finally, align the skin on the front of the neck and suture it horizontally. Disinfect the skin with iodine again, observe the rat's breathing and respiratory condition, and pay attention to whether there is swelling or bleeding around the incision.

[0099] 3. Method of administration

[0100] The pneumonia mixture pre-administration group was given pneumonia mixture gavage at a dose of 5.4 ml / kg (clinical dose conversion), once a day, for 7 consecutive days; the other experimental groups were given normal saline gavage. The dexamethasone group was given a final intraperitoneal injection of dexamethasone (5 mg / kg). 1 h after the last gavage or intraperitoneal injection, except for the blank control group, the ALI rat model was replicated in the rats of the other experimental groups, and the blank group was replaced with normal saline instead of LPS solution. 24 h after modeling, except for the pneumonia mixture treatment group, the other experimental groups were anesthetized by intraperitoneal injection of chloral hydrate to the rats, and specimens were collected; the pneumonia mixture treatment group began to be given pneumonia mixture gavage, once a day, for 7 consecutive days. After the last gavage, the rats were anesthetized by intraperitoneal injection of chloral hydrate to collect specimens.

[0101] 4. Specimen Collection

[0102] (1) Collection of whole blood, plasma, and serum

[0103] Blood was collected from the abdominal aorta into heparin tubes and ordinary serum tubes. Part of the heparin anticoagulated whole blood was used for routine blood tests, and the rest was centrifuged (3000 rpm, 10 min) to obtain plasma and serum, which were frozen at -80°C for testing.

[0104] (2) Tissue Collection

[0105] The rats were fixed in supine position after cervical dislocation, dissected, and the heart, liver, spleen, kidney, brain, whole lung tissue and trachea were completely removed, rinsed repeatedly in physiological saline, and the liquid on the surface of each tissue was fully absorbed, weighed, and frozen in a refrigerator at -80°C, except for the whole lung tissue and trachea. For the whole lung tissue and trachea, the right main bronchus was ligated with surgical sutures (to avoid air leakage), and a soft cannula of an indwelling needle was inserted between the tracheal circular cartilages. The cannula and trachea were fixed with surgical sutures (to avoid air leakage), and the left lung was lavaged with alveolar lavage. Each time, 2 ml of pre-cooled physiological saline was slowly pushed and withdrawn. In this way, the push and withdrawal were repeated many times to suck out as much lavage fluid as possible, and repeated 3 times, a total of 6 ml, and when necessary, the lung tissue was gently squeezed with gauze to obtain alveolar lavage fluid (BALF), with a yield of about 80%, and the supernatant was obtained by centrifugation (3000 rpm, 10 min), and frozen in a refrigerator at -80°C for testing. The right lung tissue of rats was separated, and the right anterior lobe was taken for determination of lung wet and dry weight; the right posterior lobe was taken and fixed in paraformaldehyde for later use; the right middle lobe was taken and frozen in a -80°C refrigerator for testing.

[0106] 5.Indicator detection and methods

[0107] The detection results of the following indicators were analyzed using the following statistics: SPSS22.0 statistical software was used for data processing and analysis, and the data were expressed as mean±SD. The expression differences between the two groups were compared using t-test, and P<0.05 was considered to be significantly different.

[0108] (1) General status

[0109] Observe the rats' hair gloss, mental state, breathing rate, activity, diet, and weight changes.

[0110] The rats in the blank control group were in good general condition, moved freely, responded sensitively to light, sound and other stimuli, had stable breathing, and showed no hair loss or dullness. The rats in the model group gradually showed symptoms such as slow response to external stimuli, chills, reduced food and water intake, weight loss, changes in stool characteristics, messy and dry fur, poor activity, and rapid breathing with tracheal wet rales. The overall condition of the rats in the positive drug group and the pneumonia mixture pre-administration group was significantly better than that in the model group. After 7 days of continuous gavage of the pneumonia mixture treatment group, the rats had normal activities, the above-mentioned symptoms were significantly alleviated, and the overall condition was better than that of the model group, the positive drug group, and the pneumonia mixture pre-administration group.

[0111] Weight changes:

[0112] The weight of rats in each group was recorded before modeling and before sacrifice, and the weight loss ratio of rats was calculated as follows: weight change ratio = (weight before sacrifice - weight before modeling) / weight before modeling. The weight of rats changed significantly after modeling. Compared with the blank group, the weight of rats in the model group (P < 0.01), dexamethasone group (P < 0.01), pneumonia mixture pre-administration group (P < 0.01), and pneumonia mixture treatment group (P < 0.05) was significantly reduced; compared with the model group, the weight of rats in the pneumonia mixture pre-administration group (P < 0.05) and pneumonia mixture treatment group (P < 0.01) increased.

[0113] The experimental results suggest that the pneumonia mixture can improve the weight loss of rats (see Figure 2 ).

[0114] (2) Organ coefficient

[0115] Before the rats were killed, their body weights were measured and recorded. The heart, liver, spleen, kidney, brain, entire lung tissue and trachea were completely removed and weighed after dissection. Finally, the organ coefficients were calculated according to the formula (organ weight / body weight × 100%). Among them, the lung coefficient is an important indicator of pulmonary edema and is usually used to evaluate the degree of pulmonary edema.

[0116] Organs were weighed and organ coefficients were calculated. Statistical analysis showed that compared with the blank group, the organ coefficients of the model group rats, except for the lung coefficient, showed no significant changes (P>0.05); the lung index of the model group and the treatment groups increased (P<0.05), indicating that the pneumonia model was successfully established; the lung index of the pneumonia mixture pre-administration group and the pneumonia mixture treatment group was lower than that of the model group (P<0.05); there was no statistically significant difference among the treatment groups (P>0.05), indicating that the pneumonia mixture had a certain improvement effect on the organ damage induced by LPS (see Figure 3 ).

[0117] (3) Lung wet weight / dry weight (W / D) ratio

[0118] The right anterior lobe of the rat lung was removed, and the blood was washed away with saline. The surface moisture of the lung was absorbed with filter paper and the wet weight (W) was measured. The lung was placed in an oven at 80°C and dried to a constant weight. The dry weight (D) was measured again, and the ratio of the wet weight to the dry weight of the lung tissue (W / D) was calculated to evaluate the degree of edema and exudation of the lung tissue.

[0119] Compared with the blank group, the wet / dry weight ratio of the lungs of rats in the model group was significantly increased (P<0.05), indicating that vascular permeability increased, the degree of pulmonary edema intensified, and the model was successfully established; compared with the model group, the dexamethasone group, the pneumonia mixture pre-administration group, and the pneumonia mixture treatment group could improve the state of pulmonary edema, and the wet / dry weight ratio of the lungs of rats was significantly reduced (P<0.01) (see Figure 4 ).

[0120] The experimental results show that pneumonia mixture can improve pulmonary edema and reduce the wet / dry weight ratio of the lungs.

[0121] (4) Pathological changes

[0122] The gross changes of lung tissue were observed with the naked eye; HE staining was used to evaluate the pathological changes of lung tissue. The posterior lobe of the right lung of the rat was fixed in 4% formalin for 3 days, and then the sample was taken, embedded in conventional paraffin, and sliced ​​into 3 μm sections. After dewaxing and rehydration, HE staining was performed, and the sections were sealed. The pathological changes of lung tissue were evaluated under an optical microscope.

[0123] i: Observe the gross changes in lung tissue with the naked eye.

[0124] The lungs of rats in the blank control group were moist, tender red, smooth, glossy, soft, elastic, with intact capsule and sharp edges. No congestion, edema or necrosis was observed. The lungs of rats in the model group were light red, with poor gloss, increased volume, obvious congestion and edema, and obvious bleeding spots and necrosis. The color of the lung tissue of rats in the positive drug group, the pneumonia mixture pre-administration group, and the pneumonia mixture treatment group was restored, the bleeding spots and necrotic foci were reduced, and the edema and congestion were significantly reduced compared with the model group (see Figure 5 ).

[0125] ii. Lung tissue pathological sections (HE staining)

[0126] The alveolar and bronchial structures of the rats in the blank control group were intact, clear, and normal in morphology, without pathological changes such as bleeding, alveolar wall thickening, and inflammatory cell infiltration. The rats in the model group had tissue lesions, damaged alveolar structures, capillary congestion, diffuse alveolar septal thickening, a large number of inflammatory cells and red blood cells infiltrated in the alveoli and pulmonary interstitium, structural disorder, partial alveolar fusion, and congestion and edema. The pathological changes in the positive drug group, the pneumonia mixture pre-administration group, and the pneumonia mixture treatment group were effectively controlled to varying degrees compared with the model group, capillary congestion was reduced, the thickening of some alveolar septa was improved, and the inflammatory cell infiltration and edema were improved (see Figure 5 ).

[0127] (5) Blood routine

[0128] Whole blood was collected in heparin tubes, and the rat whole blood red blood cell system, white blood cell system, and platelet system cells were classified and counted using a fully automatic blood cell analyzer.

[0129] i: Effects of pneumonia mixture on red blood cell parameters in rats with pneumonia.

[0130] After tracheal instillation of LPS, the hemoglobin (HGB) and hematocrit (HCT) of the model group rats increased significantly (P < 0.05), and the other parameters did not change significantly; compared with the model group, the red blood cell parameters of the pneumonia mixture pre-administration group did not change significantly, the red blood cell (RBC, P < 0.05), hemoglobin (HGB, P < 0.01), and hematocrit (HCT, P < 0.01) of the rats in the pneumonia mixture treatment group were significantly reduced, and the coefficient of variation of the red blood cell distribution width (RDW-CV) was significantly increased (P < 0.01). The experimental results suggest that LPS has no significant effect on the red blood cell parameters of rats (see Figure 6 ).

[0131] ii. Effect of pneumonia mixture on the white blood cell classification count in rats with pneumonia.

[0132] Compared with the rats in the blank group, the number of WBC in the model group was significantly decreased (P < 0.05), indicating that the body's immune function was disordered. Compared with the WBC values ​​in the model group, the number of WBC in the rats in the pneumonia mixture pre-administration group and the pneumonia mixture treatment group increased, but there was no significant difference (P > 0.05); the dexamethasone group could significantly increase the number of WBC (P < 0.05) and adjust it to normal levels, indicating that both pneumonia mixture and dexamethasone can effectively regulate the immune response in rats.

[0133] Compared with the rats in the blank group, the percentage of lymphocytes (Lym%) in the model group decreased (P < 0.01), and the percentage of intermediate cells (Mon%) and neutrophils (Neu%) increased (P < 0.01); compared with the lymphocyte values ​​of the rats in the model group, the percentage of lymphocytes (Lym%) in the rats in the pneumonia mixture pre-administration group increased, but there was no significant difference (P > 0.05); the percentage of lymphocytes (Lym%) in the rats in the pneumonia mixture treatment group increased significantly (P < 0.01). Compared with the values ​​of the model group, there were no significant changes in Mon, Mon%, Neu, and Neu% in the rats in the pneumonia mixture pre-administration group (P > 0.05); Neu% in the rats in the pneumonia mixture treatment group decreased significantly (P < 0.01) (see Figure 7 ). Therefore, the pneumonia mixture has a better effect on regulating the number of rat lymphocytes than the positive control drug (dexamethasone).

[0134] iii. Effect of pneumonia mixture on platelet parameters in rats with pneumonia.

[0135] The platelet count (PLT) and platelet volume ratio (PCT) in the peripheral blood of rats in the tracheal instillation LPS model group were significantly decreased (P < 0.05); the platelet count (PLT) and platelet volume ratio (PCT) in the pneumonia mixture pre-administration group were slightly increased, but there was no difference with the model group; the platelet count (PLT) and platelet volume ratio (PCT) in the pneumonia mixture treatment group were significantly increased (P < 0.01); there was no significant change in the mean platelet volume (MPV) among the groups; compared with the blank group, the platelet volume distribution width (PDW) of the model group rats was not significantly changed; compared with the model group value, the PDW of the pneumonia mixture treatment group rats was significantly reduced (P < 0.01) (see Figure 8 ).

[0136] (6) Biochemical indicators

[0137] According to the kit instructions, use an ELISA instrument to measure TNF-α, IL-1β, MDA, protein content (Pr), MPO activity in alveolar perfusate and Cr content in serum to examine the degree of oxidative stress damage, inflammatory response level and safety of the body. Specific kit information is as follows:

[0138] Malondialdehyde (MDA) content detection kit (Batch No.: 20220105) was purchased from Beijing Solebow Technology Co., Ltd.;

[0139] Rat TNF-α ELISA kit (lot number: J3W1Y135P9), BCA protein concentration assay kit (lot number: B2662ED2RX), and myeloperoxidase (MPO) colorimetric test kit (lot number: JPQ4PV8FP7) were purchased from Wuhan Elaruite Biotechnology Co., Ltd.;

[0140] IL-1β ELISA kit (lot number: 11517126119, Boster Biotechnology Co., Ltd.);

[0141] Creatinine (Cr) assay kit (Batch No.: 20220406, Nanjing Jiancheng Bioengineering Institute).

[0142] After LPS induction, the contents of TNF-α and IL-1β in BALF of rats in the model group, pneumonia mixture pre-administration group and dexamethasone group were significantly increased (P<0.01). There were no significant differences in the contents of TNF-α and IL-1β in BALF between the pneumonia mixture treatment group and the blank group after 8-day oral administration (P>0.05). Compared with the model group, the contents of TNF-α and IL-1β in BALF of rats in the pneumonia mixture pre-administration group, pneumonia mixture treatment group and dexamethasone group were significantly decreased (P<0.01). The differences in TNF-α and IL-1β in BALF of rats in the pneumonia mixture treatment group and those in the pneumonia mixture pre-administration group, and between the dexamethasone group and the pneumonia mixture treatment group were statistically significant (P<0.01). The difference in TNF-α in BALF of rats in the dexamethasone group and those in the pneumonia mixture pre-administration group was statistically significant (P<0.01).

[0143] After LPS induction, the protein content in BALF of rats in the model group was significantly increased (P<0.01), and the vascular permeability was increased, showing an acute inflammatory response, indicating that the modeling was ideal; the protein content in BALF of rats in the pneumonia mixture pre-administration group and dexamethasone group was significantly increased (P<0.01); there was no significant difference in the protein content in BALF between the pneumonia mixture treatment group and the blank group after 8 days of oral administration (P>0.05); compared with the model group, the protein content in BALF of rats in the pneumonia mixture treatment group was significantly decreased (P<0.01), while the protein content in BALF of rats in the pneumonia mixture pre-administration group and dexamethasone group was decreased, but the difference was not statistically significant (P>0.05).

[0144] Compared with the blank group, the MPO activity in BALF of the model group was increased, but there was no significant difference (P>0.05); compared with the model group, the MPO activity in BALF of rats in the pneumonia mixture pre-administration group and the pneumonia mixture treatment group was decreased, but there was no significant difference (P>0.05).

[0145] Compared with the blank group, the MDA level in bronchoalveolar lavage fluid of the model group was increased (P<0.05); compared with the model group, the MDA level in BALF of rats in the pneumonia mixture pre-administration group, pneumonia mixture treatment group, and dexamethasone group was decreased, but there was no significant difference (P>0.05).

[0146] There was no significant difference in serum creatinine content among the blank group, model group, pneumonia mixture pre-administration group, and pneumonia mixture treatment group (P>0.05) (see Fig. 9 ).

[0147] In the above embodiments, Figure 2 , Figure 3 , Figure 4 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 Compared with the normal group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the pneumonia mixture pre-administration group, & P<0.05, && P<0.01; compared with the pneumonia mixture treatment group, ￡ P<0.05, ￡￡ P<0.01.

[0148] Example 4

[0149] This example conducts an acute toxicity test study on the pneumonia mixture.

[0150] 1. Drug preparation

[0151] The pneumonia mixture was placed in a 60°C water bath to be concentrated into a fluid extract (relative density of about 1.20 (60°C)), and dried under reduced pressure at -0.085MPa and 70°C to prepare a dry extract powder of the pneumonia mixture, which was placed in a desiccator for standby use. The dry extract yield was about 30%. When used, distilled water was used to prepare the maximum mass concentration (the maximum concentration that can pass through a No. 12 mouse gavage needle), and the concentration was calculated as 0.99g / ml (10.5g compound crude drug / mL) based on the extract concentration, and stored in a 4°C refrigerator for standby use.

[0152] 2. Maximum Dosage Experiment

[0153] Twenty ICR mice were taken and divided into two groups, a blank group and a pneumonia mixture group, with 10 mice in each group. They were fasted but not watered for 12 hours before the experiment. The drug-treated group was gavaged twice with the maximum mass concentration of the extract of 0.99g / ml, the gavage volume was 20mL / kg, the interval between the two times was 6h, and the total dose was 39.6g / kg; the blank group was gavaged with an equal volume of distilled water. After administration, the mice were fed routinely, and the toxic reactions and deaths of the mice and the spirit, eating, drinking, breathing, death, fur, feces and movement of the experimental mice were observed. The weight of the mice was recorded every other day, and the observation was continued for 14 days. After 14 days, blood was collected from the eye sockets, and the blood was centrifuged at 3000rpm for 10 minutes after standing. The serum was used for the determination of blood biochemical indicators ALT, AST, BUN, and Cr, and placed in a -80℃ refrigerator for detection. The mice were killed and autopsied to observe whether there were visual pathological changes in the solid organs of the mice, and the weight of the heart, liver, spleen, lung, and kidney was measured and the organ coefficient was calculated.

[0154] i. General performance results of animals in the acute toxicity test of pneumonia mixture:

[0155] After intragastric administration, the mice were in good condition, with no abnormalities in behavior, mental state, diet, fur gloss, defecation, urination, and movement. There was no secretion from the anus, nose, eyes, or mouth, and no other toxic reactions occurred. No animal died. After the animals were killed, no obvious changes were observed in the morphology of the organs (see Fig.10 ).

[0156] ii. Weight changes

[0157] There was no significant difference in body weight between the treated group and the control group (P>0.05) (see Fig.11 ).

[0158] iii. Organ coefficient

[0159] The organ coefficients were calculated by weighing the organs. Statistical analysis showed that the organ coefficients of the liver, spleen, lung and kidney of the two groups of mice had no significant difference (P>0.05), except for the significant difference in the organ coefficient of the female group (P<0.05). Fig.12 ). Fig.12 Compared with the control group, * P<0.05.

[0160] iv. Blood biochemical indicators

[0161] The operation was strictly in accordance with the instructions of the kit, and the detection was performed by ELISA. There was no significant difference in blood biochemical indicators ALT, AST, BUN, and creatinine between the two groups (P>0.05) (see Fig.13 ).

[0162] In summary, the present invention conducts chemical component analysis on pneumonia mixture: based on UHPLC-QE-MS high-resolution liquid-mass spectrometry technology combined with BiotreeDB (V2.1) secondary mass spectrometry database matching screening platform, an effective method for rapid analysis and identification of the chemical components of pneumonia mixture is established, and a total of 123 compounds, including alkaloids, flavonoids and other components, are found in positive and negative ion modes through comparison with reference substances, comparison with literature mass spectrometry fragment data, and matching with mass spectrometry database. The results can provide a reference for strengthening the overall quality control of pneumonia mixture and preliminarily clarifying the material basis of efficacy, and provide data support for subsequent studies on the metabolism and mechanism of action of the components of pneumonia mixture.

[0163] The results of the pharmacodynamic study of pneumonia mixture showed that the development of inflammation in the lungs of rats after LPS induction had a certain regularity, and after treatment, the inflammation was alleviated. Dexamethasone has an ameliorative effect on the lung injury of the ALI rat model induced by LPS, and both pre-administration and treatment administration of pneumonia mixture can also play a protective role in rats with lung injury by directly regulating inflammation and other mechanisms, effectively controlling the development of inflammation, reducing the level of inflammation, reducing lung oxidative damage, and improving the expression of related inflammatory factors, playing a positive role in relieving lung inflammation, providing a reference for the clinical application of pneumonia mixture in the treatment of pediatric pneumonia.

[0164] The results of the acute toxicity test on pneumonia mixture showed that pneumonia mixture had no obvious acute toxicity, was safe and reliable for oral administration, had a wide safety range, and had good clinical application safety.

[0165] In conclusion, pneumonia mixture can be used for the prevention and treatment of acute lung injury, with good efficacy, small toxic and side effects, and has good prospect value.

[0166] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a composition in the preparation of a drug for preventing or treating acute lung injury, characterized in that: The composition comprises the following raw materials in percentage by weight: 5-20% of scutellaria baicalensis, 1-15% of liquorice, 1-10% of ephedra, 10-25% of isatis root, 10-20% of gypsum, 5-15% of bitter almond, 15-25% of reed root and 10-20% of honeysuckle. The preparation method of the composition comprises: extracting the raw materials with water, concentrating the filtrate obtained by filtration, then precipitating the concentrated solution with alcohol, and collecting the ethanol solution obtained by the alcohol precipitation.

2. The use according to claim 1, characterized in that: The drug has at least one of the following uses: (1) Improve the weight loss of subjects; (2) Improvement of organ damage in subjects; (3) improving the subject's pulmonary edema and / or congestion state; (4) reduce the wet / dry weight ratio of the subjects' lungs; (5) restore the color of the subject's lung tissue; (6) Reduce the bleeding points and necrotic lesions in the subjects' lung tissue; (7) reducing capillary congestion in the subject's lung tissue; (8) improving alveolar septal thickening in the subject's lung tissue; (9) improving inflammatory cell infiltration in the lung tissue of the subject; (10) Improve the abnormal blood routine test results of the subjects; (11) Improve the abnormal biochemical indicators of the subjects.

3. The use according to claim 2, characterized in that: The improvement of the abnormal blood routine of the subject includes at least one of the following methods: (1) Improve the subject's red blood cell abnormalities; (2) improving the subject's leukocyte abnormalities; (3) Improve platelet abnormalities in subjects.

4. The use according to claim 3, characterized in that: The improvement of the abnormal blood routine of the subject includes at least one of the following methods: (1) Reduce hemoglobin (HGB) level, hematocrit (HCT), neutrophil count (Neu) or ratio, intermediate cell count (Mon) or ratio, and platelet distribution width (PDW); (2) Increased white blood cell (WBC) count or ratio, lymphocyte (Lym) count or ratio, platelet (PLT) count or ratio, and platelet count (PCT).

5. The use according to claim 3, characterized in that: The improvement of the abnormal blood routine of the subject includes the following methods: Reduce the levels of TNF-α, IL-1β, protein, malondialdehyde (MDA) and myeloperoxidase (MPO) activity in bronchoalveolar lavage fluid.

6. The use according to claim 3, characterized in that: The subject is a human or a non-human mammal.

7. The use according to any one of claims 1 to 6, characterized in that: The acute lung injury was induced by LPS.

8. The use according to any one of claims 1 to 6, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

9. The use according to claim 8, characterized in that: The pharmaceutically acceptable carrier is selected from one or more of starch, pregelatinized starch, microcrystalline cellulose, lactose, lactose starch complex, lactose cellulose complex, mannitol, mannitol starch complex, sorbitol, povidone, copovidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, magnesium stearate, sodium stearyl fumarate, talc, and stearic acid.

10. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug is selected from any one of an oral preparation and an injection.

Images