WO2021179828A1

WO2021179828A1 - Use of pharmaceutical composition in preparation of drug for treating acute lung injury

Info

Publication number: WO2021179828A1
Application number: PCT/CN2021/074021
Authority: WO
Inventors: 贾振华
Original assignee: 石家庄以岭药业股份有限公司
Priority date: 2020-03-11
Filing date: 2021-01-28
Publication date: 2021-09-16
Also published as: CN113384647B; CN113384647A

Abstract

Disclosed is the use of a pharmaceutical composition in the preparation of a drug for treating acute lung injury. The pharmaceutical composition is prepared from Ephedrae herba, Gypsum fibrosum, Forsythiae fructus, Scutellariae radix, Mori cortex, Armeniacae semen amarum, Peucedani radix, Pinelliae rhizoma, Citri reticulatae pericarpium, Fritillaria, Arctii fructus, Lonicerae japonicae flos, Rhei radix et rhizoma, Platycodonis radix, and Glycyrrhizae radix et rhizoma.

Description

[Name of invention formulated by ISA according to Rule 37.2] "Application of a pharmaceutical composition in the preparation of drugs for the treatment of acute lung injury

This application claims the priority of the Chinese patent application 202010164927.4 filed to the Chinese Patent Office on March 11, 2020 with the title "Application of a Chinese Medicine Composition in the Preparation of Medicines for the Treatment of Acute Lung Injury", the content of which is incorporated by reference The whole is incorporated into this article.

Technical field

The invention relates to the application of a traditional Chinese medicine composition in the preparation of a medicine for treating acute lung injury, and belongs to the field of medicine.

Background technique

Acute lung injury (ALI) is a common clinical critical illness. The main factors that cause acute lung injury are systemic infection, sepsis, trauma, shock, poisoning and so on. The main pathological manifestations include damage to pulmonary vascular endothelial cells, destruction of endothelial integrity, impaired alveolar-capillary barrier function, increased vascular permeability, extensive inflammatory cell infiltration in the lungs, and pulmonary edema. If not treated in time, acute lung injury will develop into acute respiratory distress syndrome, with a fatality rate of up to 40%. At present, the diagnosis of ALI still follows the standards proposed by the 1994 European and American Joint Conference: (1) Acute onset, there are pathogenic factors; (2) Oxygenation index (arterial blood oxygen partial pressure/inhaled oxygen concentration, PaO ₂ /FiO ₂ )<300mmHg (1mm Hg=0.133kPa), without reference to the level of positive end expiratory pressure (PEEP); (3) An orthographic chest radiograph showed patchy shadows in both lungs; (4) Pulmonary incarceration pressure <18mmHg, or no clinical evidence of increased left atrial pressure; (5) Acute onset respiratory failure.

The currently recommended treatment for acute lung injury is comprehensive treatment, which not only corrects refractory hypoxemia and improves alveolar oxygenation, but also a comprehensive treatment centered on appropriate respiratory support techniques, including infection control and early nutritional support treatment. , Prevention of stress ulcers, prevention of deep vein thrombosis and many other adjuvant treatments, even including delicate care, currently there is no specific drug treatment. In the existing treatments, symptomatic and supportive treatment occupies an important position. However, although symptomatic and supportive treatment can alleviate clinical symptoms, it cannot obviously control the progress of the disease and has certain limitations. Therefore, there is an urgent need to provide an effective and safe treatment for acute lung injury in the prior art.

Summary of the invention

The inventors unexpectedly discovered that the pharmaceutical composition of the present invention can effectively treat acute lung injury. The pharmaceutical composition of the present invention and its preparation method were first disclosed in CN101549060A, and the contents recorded in the document are quoted here in full. The inventor unexpectedly discovered in the research that the pharmaceutical composition has a particularly good therapeutic effect on acute lung injury.

Based on this discovery, in the first aspect, the present invention provides an application of a pharmaceutical composition in the preparation of a medicine for the treatment of acute lung injury. The active ingredient of the pharmaceutical composition is made of the following raw materials by weight:

Ephedra 52-86; Gypsum 194-324; Forsythia 194-324; Scutellaria 78-130; Mulberry Bark 194-324; Bitter Almond 78-130; Qianhu 78-130; Pinellia 78-130; Tangerine Peel 78-130 Fritillaria 78-130; Arctium lappa 78-130; Honeysuckle 78-130; Rhubarb 39-65; Platycodon 46-76; Licorice 39-65.

The weight ratio of the bulk drug of the pharmaceutical composition of the present invention is preferably:

Ephedra 52; Gypsum 324; Forsythia 194; Scutellaria 78; Mulberry 194; Bitter Almond 130; Qianhu 78; Pinellia 130; Tangerine Peel 78; Fritillaria 78; Burdock Seed 130; Honeysuckle 130; Rhubarb 39; Platycodon 76; Licorice 65.

The weight ratio of the bulk drug of the pharmaceutical composition of the present invention is also preferably:

Ephedra 86; Gypsum 194; Forsythia 324; Scutellaria 130; Mulberry Bark 324; Bitter Almond 78; Qianhu 130; Pinellia 78; Tangerine Peel 130; Fritillaria 130; Burdock 78; Honeysuckle 78; Rhubarb 65; Platycodon 46; Licorice 39.

Ephedra 69; Gypsum 259; Forsythia 259; Scutellaria baicalensis 104; Mulberry bark 259; Bitter almond 104; Qianhu 104; Pinellia 104; Licorice 52.

Ephedra 55; Gypsum 254; Forsythia 318; Scutellaria baicalensis 107; Mulberry bark 203; Bitter almond 107; Qianhu 82; Pinellia 105; Tangerine peel 84; Fritillaria 125; Burdock 122; Honeysuckle 113; Rhubarb 42; Licorice 50.

In the pharmaceutical composition of the present invention, preferably, the bitter almonds are fried bitter almonds, the fritillary is Fritillaria fritillary, the honeysuckle is the Lonicera japonica, and the Pinellia ternata is the Qing Pinellia.

In the second aspect, the application of the pharmaceutical composition of the present invention in the preparation of a medicament for the treatment of acute lung injury is provided, wherein the active ingredient of the pharmaceutical composition is prepared by a method including the following steps:

A. Take the mother-of-pearl and crush it;

B. Extract ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb with solvent and concentrate to obtain a concentrate;

C. Extract gypsum, mulberry bark, Peucedanum praeruptorum, tangerine peel, mountain silver flower, platycodon, and licorice with solvent and concentrate to obtain a concentrate;

D. Combine the concentrate of step B and the concentrate of step C, and add the powder obtained in step A to the concentrate to obtain the pharmaceutical composition of the present invention.

Preferably, Fritillaria is pulverized into fine powder in step A; preferably, the solvent used for extraction in step B is an organic solvent, preferably an alcohol, particularly preferably a C1-C4 alkanol, and particularly preferably ethanol; preferably Specifically, the solvent used for extraction in step C is water.

In steps B and C, the solvent extraction is preferably extracted twice, and the two extracts are combined; the extraction method is preferably reflux extraction; the amount of solvent used is preferably 8-11 times the amount of solvent used in the first extraction (based on the weight of the medicinal material) , The second extraction uses 6-9 times the amount of solvent; the extraction time is preferably 1-4 hours each time.

In steps B and C, it is preferably concentrated to the extent of a clear ointment with a relative density of 1.14-1.16 as measured at 60°C.

In a particularly preferred embodiment of the present invention, the preparation method of the pharmaceutical composition of the present invention includes the following steps:

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, Qing Pinellia, burdock seeds, rhubarb, and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 for the first time. Double the amount, add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C and heat the clear cream with a relative density of 1.14-1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, tangerine peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportions of the prescriptions. Add water and decoct twice for 1-4 hours each time, and add 9-11 times the amount for the first time. Add 7-9 times the amount for the second time, combine the decoction, filter, concentrate to 60°C and measure the clear ointment with a relative density of 1.14-1.16, and combine with the clear ointment obtained in step B for use;

D. Mix the fine powder obtained in step A and the combined clear ointment obtained in step C to obtain the pharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention prepared according to the above method can also be added with suitable carriers and/or excipients to prepare the pharmaceutical composition of the present invention suitable for various administration routes and dosage forms.

The medicine of the present invention may be in the form of capsules, tablets, powders, granules, oral liquids, soft capsules, pills, tinctures, syrups, suppositories, gels, sprays or injections.

In order to realize the above dosage forms, it is necessary to add pharmaceutically acceptable excipients when preparing these dosage forms, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, correctives, and preservatives. , Substrate, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, Cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agent Including: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: Sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; correctives include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and other Salts, benzalkonium bromide, chloroethyl acetate, eucalyptus oil, etc.; bases include: PEG6000, PEG4000, insect wax, etc.

In a preferred embodiment of the present invention, the tablet is made by the following steps:

C. Weigh gypsum, Morus alba peel, Peucedanum, tangerine peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportions of the prescriptions. Add water and decoct twice for 1-4 hours each time, and add 9-11 times the amount for the first time. Add 7-9 times the amount for the second time, combine the decoction, filter, and concentrate to 60°C to measure the clear ointment with a relative density of 1.14-1.16, combine with the clear ointment obtained in step B, and set aside;

D. Spray dry the combined cleansing paste obtained in step C, and collect the spray powder for later use;

E. The spray powder obtained in step D and the fine powder obtained in step A are prepared by using ethanol as a binder to prepare a soft material, and then sieving to granulate; it is obtained by pressing according to the conventional method of pharmacy.

In a more preferred embodiment, the tablet of the present invention is made by a method comprising the following steps:

B. Weigh ephedra, forsythia, sauteed bitter almonds, Qing Pinellia, burdock seed, rhubarb and reflux with 50% ethanol for 2 times, 3 hours each time, 10 times the amount for the first time, and the second time Add 6 times the amount, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C and heat the clear cream with a relative density of 1.15 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription. Add water and decoct twice for 2 hours each time. Add 10 times the amount for the first time and add it for the second time 7 times the amount, the decoction is combined, filtered, concentrated to 60°C, the clear ointment with a relative density of 1.15, and the clear ointment obtained in step B is combined and set aside;

E. The spray powder obtained in step D and the fine powder obtained in step A are prepared with ethanol as a binder to make a soft material, sieved, granulated and dried, and then sieved. Add sodium starch glycolate, microcrystalline cellulose, and magnesium stearate, and mix well , Compressed tablets are available.

An exemplary preparation method for other dosage forms of the drug of the present invention is: weighing the bulk drug in proportion and preparing it by a conventional preparation method, for example, the preparation described in Fan Biting's "Chinese Medicine Pharmacy" (Shanghai Science Press, December 1997, 1st edition) Process, made into a pharmacologically acceptable conventional dosage form.

The pharmaceutical composition of the present invention can also reduce the increase of cytoinflammatory factors caused by acute lung injury.

The pharmaceutical composition of the present invention can reduce cytoinflammatory factors caused by acute lung injury, and the cytoinflammatory factors can be IL-6, TNF-α, NE, and MCP-1.

The pharmaceutical composition of the present invention can also inhibit the signal transduction of protein expression caused by acute lung injury.

The pharmaceutical composition of the present invention can inhibit the protein caused by acute lung injury, and the protein can be NF-κB P65, IκB-α, p-IκB-α, and IKKβ.

Description of the drawings:

Figure 1: Shows the effect of the pharmaceutical composition of the present invention on the expression of NF-κB P65, IκB-α, p-IκB-α, IKKβ protein in acute lung injury lungs, where A is the normal control group and B is the model control group , CE is the low-dose group, middle-dose group and high-dose group of the pharmaceutical composition of the present invention.

Figure 2: shows the HE staining observation of the lungs of acute lung injury model mice with the pharmaceutical composition of the present invention (×100), where A is the normal control group, B is the model control group, and CE is the drug composition of the present invention. Low-dose group, middle-dose group and high-dose group.

Detailed ways

The following examples are used to illustrate the preparation of the pharmaceutical composition of the present invention, but should not be construed as limiting the protection scope of the present invention.

In the following examples, the prescriptions are all the amount of raw materials required to make 1000 preparation units (tablets, capsules, etc.).

Preparation Example 1

prescription:

Ephedra 52g; gypsum 324g; forsythia 194g; skullcap 78g; mulberry 194g; bitter almond 130g; Qianhu 78g; pinellia 130g; tangerine peel 78g; zhejiang fritillary 78g; burdock seed 130 grams; 130 grams of licheniflora; 39 grams of rhubarb; 76 grams of platycodon; 65 grams of licorice. Preparation:

B. Weigh ephedra, forsythia, bitter almond, pinellia, burdock, and rhubarb with 50% ethanol refluxing extraction 2 times, 3 hours each time, 10 times the amount for the first time, and 6 times for the second time. Double the volume, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure a clear paste with a relative density of 1.15 for use;

E. The spray powder obtained in step D, the fine powder obtained in step A, prepare a soft material with 80% ethanol as a binder, sieving and granulating, and drying at 60°C before sizing. Add sodium carboxymethyl starch, microcrystalline cellulose, and magnesium stearate, mix well, and prepare tablets according to the conventional preparation method to obtain.

Preparation Example 2

prescription:

Ephedra 86g; Gypsum 194g; Forsythia 324g; Scutellaria baicalensis Georgi 130g; Mulberry bark 324g; Fried bitter almond 78g; Qianhu 130g; Pinellia ternata 78g; Tangerine peel 130g; Fritillaria 130g; Burdock seed 78 grams; 78 grams of Lonicera japonica; 65 grams of rhubarb; 46 grams of platycodon; 39 grams of licorice. Preparation:

A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, pinellia, burdock seeds, rhubarb and reflux with 40% ethanol for 2 times, 4 hours each time, add 8 times the amount for the first time, and add it for the second time. 9 times the amount, the extracts are combined, filtered, the filtrate is decompressed to recover ethanol, and concentrated to a clear paste with a relative density of 1.14 at 60°C for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescriptions. Add water and decoct twice for 4 hours each time. Add 9 times the amount for the first time and add it for the second time 7 times the amount, the decoction is combined, filtered, concentrated to 60°C and the clear ointment with a relative density of 1.16, which is thermally measured, is combined with the clear ointment obtained in step B for use;

E. The spray powder obtained in step D, the fine powder obtained in step A, use 80% ethanol as a binder to prepare a soft material, sieving and granulating, drying at 60 degrees, and then sizing. Add sodium carboxymethyl starch, microcrystalline cellulose, and magnesium stearate, mix well, and prepare tablets according to the conventional preparation method to obtain.

Preparation Example 3

prescription:

Ephedra 69g; Gypsum 259g; Forsythia 259g; Scutellaria baicalensis Georgi 104g; Mulberry bark 259g; Fried bitter almonds 104g; Qianhu 104g; Qing Pinellia 104g; Tangerine peel 104g; Zhejiang Fritillaria 104g; 104 grams of burdock seeds; 104 grams of lichen; 52 grams of rhubarb; 61 grams of platycodon; 52 grams of licorice.

Preparation:

B. Weigh ephedra, forsythia, sauteed bitter almonds, Qing Pinellia, burdock seeds, rhubarb, and reflux with 70% ethanol for 2 times, 1 hour each time, 10 times the amount of the first time, and the second time Add 6 times the amount, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure a clear paste with a relative density of 1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, Chenpi, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription, add water and decoct twice for 1 hour each time, add 11 times the amount for the first time, and add it for the second time. 7 times the amount, the decoction is combined, filtered, concentrated to 60°C, the clear ointment with a relative density of 1.14, and the clear ointment obtained in step B is combined and set aside;

Preparation Example 4:

The API formula is:

Ephedra 55g; Gypsum 254g; Forsythia 318g; Scutellaria baicalensis 107g; Mulberry bark 203g; Fried bitter almonds 107g; Qianhu 82g; Pinellia ternata 105g; Chenpi 84g; 122 grams of seeds; 113 grams of lichen; 42 grams of rhubarb; 60 grams of platycodon; 50 grams of licorice.

Preparation:

B. Weigh ephedra, forsythia, sauteed bitter almonds, pinellia, burdock seeds, rhubarb, and reflux with 60% ethanol for 2 times, 2 hours each time, add 9 times the amount for the first time, and add it for the second time. 7 times the amount, the extracts are combined, filtered, and the filtrate is reduced to recover ethanol, and concentrated to 60°C to measure a clear ointment with a relative density of 1.15 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription, add water and decoct twice for 2.5 hours each time, add 10 times the amount for the first time, and add it for the second time. 7 times the amount, the decoction is combined, filtered, concentrated to 60°C, the clear ointment with a relative density of 1.14, and the clear ointment obtained in step B is combined and set aside;

Preparation Example 5:

Ephedra 62g Plaster 220g Forsythia 256g Radix Scutellaria 90g Mulberry Bark 300g Bitter Almond 90g Qian Hu 90g Pinellia 90g Tangerine Peel 100g Fritillaria 100g Burdock Seed 100g Honeysuckle 100g Rhubarb 50g Platycodon 66g Licorice 50g

The above medicinal materials can be obtained as capsules according to conventional methods.

Preparation Example 6:

Ephedra 68g Plaster 215g Forsythia 215g Radix Scutellariae 100g Mulberry Bark 220g Bitter Almond 90g Qian Hu 90g Pinellia 90g Tangerine Peel 90g Fritillaria 90g Burdock Seed 90g Honeysuckle 90g Rhubarb 50g Platycodon 50g Licorice 50g

The above medicinal materials are prepared into granules according to conventional methods.

Preparation Example 7:

Ephedra 50g Plaster 200g Forsythia 300g Scutellaria baicalensis 100g Mulberry bark 250g Bitter almond 100g Qianhu 100g Pinellia 100g Tangerine peel 100g Fritillaria 100g Burdock seed 100g Honeysuckle 100g Rhubarb 50g Platycodon 50g Licorice 50g

The above medicinal materials can be obtained as injections according to conventional methods.

Preparation Example 8:

Ephedra 60 g Plaster 200 g Forsythia suspense 200 g Scutellaria baicalensis Georgi 95 g Mulberry bark 230 g Bitter almond 95 g Qianhu 95 g Pinellia 95 g Tangerine peel 95 g Fritillaria 95 g Burdock seed 95 g Honeysuckle 95 g Rhubarb 50 g Platycodon 50 g Licorice 50g

The above medicinal materials can be obtained as pills according to conventional methods.

Experimental example:

In order to verify that the pharmaceutical composition of the present invention has the effect of treating acute lung injury, the following pharmacological test studies were carried out using the tablets prepared according to the method of Preparation Example 3 (hereinafter referred to as the drug of the present invention):

Materials and Methods

1. Experimental materials

1.1 Medicines

1.1.1 Test product

The pharmaceutical composition of the present invention has a specification: 1.843 g crude drug/tablet, provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd.

1.1.2 Solvent

Distilled water, homemade.

1.2 Experimental animals

60 BALB/c mice, SPF grade, half male and half male, weighing 17.6～23.4g, the laboratory animal quality certificate provided by Hunan Slack Jingda Biotechnology Co., Ltd.: No.43004700050920, the laboratory animal production license number: SCXK (Hunan) 2016-0002; Raised in Zone A of the barrier of the Hunan Drug Safety Evaluation and Research Center, laboratory animal license number: SYXK (Hunan) 2015-0016.

1.3 Main reagents

Lipopolysaccharide (LPS, product number: L2880, product of Sigma), mouse IL-6 ELISA kit (lot number: 201811), mouse TNF-α ELISA kit (lot number: 201811), mouse neutrophil elastase ( NE) ELISA kit (lot number: 201811), mouse SOD ELISA kit (lot number: 201811), mouse MDA ELISA kit (lot number: 201811), mouse NO ELISA kit (lot number: 201811), mouse single Nuclear cell chemoattractant protein-1 (MCP-1) ELISA kit (lot number: 201811), the above kits are all Bio-Swamp products; human nuclear factor kappa B inhibitory protein alpha (IKB-α) primary antibody (catalog number: ab32518) ), phosphorylated human nuclear factor kappa B inhibitor protein alpha (p-IKB-α) primary antibody (Cat. No.: ab133462), human nuclear factor kappa B inhibitor kinase beta (IKK-β) primary antibody (Cat. No.: ab124957), above Antibodies are all products of the British company abcam; nuclear factor κB P65 (NF-κB P65) primary antibody (Cat. No.: 10745-1-AP), actin (β-actin) primary antibody (Cat. No.: 60008-1-Ig) ), the above antibodies are all products of American proteintech company.

1.4 Main instruments

Spectra Max i3x multifunctional microplate reader (manufactured by Miguguang, Austria); DFC 420C pathological imaging system (manufactured by Leica, Germany); JY04S-3C gel imaging analysis system (manufactured by Beijing Junyi Dongfang Company).

2. Experimental method

2.1 Modeling, grouping and drug delivery

60 BALB/c mice, SPF grade, half male and half male, weighing 17.6-23.4g, randomly divided into normal control group, model control group, low, medium and high dose groups of the pharmaceutical composition of the present invention (corresponding to 1.83, 3.67, 7.33g crude drug/kg) dose group, each group has 10 animals. Before daily administration, the pharmaceutical composition of the present invention (grinded into powder) is formulated with distilled water to prepare a corresponding concentration of liquid. Mice in each group were given the corresponding medicinal solution by gavage at 20 mL/kg, once a day, for 7 consecutive days, and the normal control group and the model control group were gavage with equal volume of distilled water. One hour after the last administration, mice in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate 300mg/kg. The mice were fixed on a surgical board, the neck was disinfected, the neck skin was cut longitudinally, the subcutaneous tissue was stripped, and the trachea was exposed; Except for the control group of the normal group, mice in the other groups were injected with lipopolysaccharide into the trachea at 0.5 mg/kg to induce acute lung injury model. Immediately after the injection, the mice were erected and the wounds were sutured after shaking slightly to distribute the liquid evenly in the lungs. The normal control group was injected with the same dose of 0.9% sodium chloride injection, and the mice were awake naturally.

2.2 Testing indicators

Mice in each group were sacrificed 5 hours after the completion of the model, the lungs were dissected and the lungs were taken out, and the left lungs were weighed (the right lungs were sampled for use) and placed in a homogenizer. According to lung mass (g): normal saline (mL) =1:9 Add 0.9% sodium chloride injection, grind and homogenize, make rat lung suspension, centrifuge at 2500rpm for 20min at 4℃, take the supernatant, ELISA kit detects MDA, SOD, NO, MCP-1 Content: Blood was collected from the orbit, and the serum content of IL-6, TNF-α, NE was detected according to the ELISA kit method; the lung tissue was dissected, and the lung tissue was taken, and WB was used to detect NF-κB P65, IκB-α, p-IκB in the lung tissue -α, IKKβ protein expression; dissected the right lung tissue, fixed in formalin, embedded in paraffin, sectioned, HE stained, observed the pathological changes of the lung under light microscope, and observed the lung injury and inflammatory cell infiltration The specific scoring criteria are as follows: 0 points: normal; 1 point: mild (lesion less than 30%); 2 points: moderate (lesion 30%-50%); 3 points: severe (lesion greater than 50%).

2.3 Dosage design

See Table 1 for specific grouping and dosage design.

Table 1 Grouping and dose design

2.4 Statistical methods

The effective figures of the test data were rounded off and statistically analyzed in accordance with SOP regulations. The software used for statistics is SPSS 16.0. Measurement data are based on mean ± standard deviation

Indicates that the Leven's test method is used to test normality and homogeneity of variance. If there is no statistical significance (P>0.05), use one-way analysis of variance (ANOVA) for statistical analysis. If ANOVA is statistically significant (P≤0.05), use LSD test (parameter method) for comparative analysis. If the variance is not uniform (P≤0.05), use the Kruskal-Wallis test. If the Kruskal-Wallis test is statistically significant (P≤0.05), Dunnett's Test (non-parametric method) is used for comparative analysis. For the statistical results, α=0.05 is the test limit, where P≤0.05 indicates statistical significance, and P≤0.01 indicates that the difference tested is very significant.

3. Results

3.1 Effect on lung antioxidant capacity of acute lung injury model

As shown in Table 2, compared with the normal control group, the MDA content in the alveolar lavage fluid of the model control group increased significantly (P≤0.01), and the SOD and NO decreased significantly (P≤0.01); compared with the model control group The MDA content in the mouse alveolar lavage fluid of the low, medium, and high dose groups of the pharmaceutical composition of the present invention was significantly reduced (P≤0.01), and the SOD content was significantly increased (P≤0.01), and the high-dose group of mice The NO content in the liquid was significantly increased (P≤0.01), suggesting that the pharmaceutical composition of the present invention can alleviate oxidative stress by reducing the deposition of lipid peroxides in the lungs of acute lung injury model mice, and can increase the content of antioxidant enzymes. To enhance the antioxidant capacity.

Table 2 The effect of the pharmaceutical composition of the present invention on the antioxidant capacity of acute lung injury mice (

n=10)

Note: Compared with the normal control group ⁺⁺ P≤0.01, compared with the model control group ^* P≤0.05, ^** P≤0.01.

3.2 Effect on the release of inflammatory factors in inflammatory cells of acute lung injury

As shown in Table 3, compared with the normal control group, the serum levels of IL-6, TNF-α, NE, and MCP-1 in the model control group increased significantly (P≤0.05 or P≤0.01); compared with the model control group In comparison, the levels of IL-6 and TNF-α in the serum of mice in the low, medium and high dose groups of the pharmaceutical composition of the present invention were significantly reduced (P≤0.05 or P≤0.01), and the levels of NE and MCP-1 in the high dose group were significantly reduced (P≤0.05), suggesting that the medicament of the present invention can significantly alleviate the inflammatory response in the lungs.

Table 3 The effect of the pharmaceutical composition of the present invention on inflammatory factors in the serum of mice with acute lung injury (

n=10)

Note: Compared with the normal control group ⁺ P≤0.05, compared with the model control group ^* P≤0.05, ^** P≤0.01.

3.3 Effect on NF-κB P65, IκB-α, p-IκB-α, IKKβ protein expression in lungs of acute lung injury model

As shown in Table 4 and Figure 1, compared with the normal control group, the expression of IκB-α, p-IκB-α, IKKβ, and NF-κB P65 in the lung tissue of the model control group increased significantly (P≤0.01 ); Compared with the model control group, the expression of IκB-α and p-IκB-α in the lungs of mice in the low, medium and high dose groups of the pharmaceutical composition of the present invention was significantly reduced (P≤0.05 or P≤0.01). The expression of IKKβ, NF-κB and P65 in the dose group was significantly reduced (P≤0.01), suggesting that the pharmaceutical composition of the present invention can inhibit the transduction of IKK/IκB/NF-κB.

Table 4 The effect of the pharmaceutical composition of the present invention on protein expression in the lungs of mice (

n=10)

3.3 Effect on the lungs of mice with acute lung injury

As shown in Table 5 and Figure 2, the alveolar structure of the mice in the normal control group is complete, no obvious congestion, no inflammatory cell infiltration, and normal alveolar wall structure. Compared with the normal control group, the pathological scores of mice in the model control group increased significantly (P≤0.01). The pathological changes were mainly manifested by the infiltration of a large number of inflammatory cells in the alveolar cavity, red blood cell exudation, obvious thickening of alveolar walls, and obvious proliferation of alveolar interstitium. Edema. Compared with the model control group, the pathological scores of the middle and high-dose groups of the pharmaceutical composition of the present invention were significantly reduced (P≤0.05 or P≤0.01). The mice in the middle-dose group of the pharmaceutical composition of the present invention had relatively complete alveolar structure and a small amount of inflammation. Sex cell infiltration, hyperplasia, and edema were significantly reduced. The alveolar results of the mice in the high-dose group were complete and all indicators were close to the normal control group.

Table 5 The effect of the pharmaceutical composition of the present invention on the pathological score of lung tissue in mice (

n=10)

4 Conclusion

The above test results show that the pharmaceutical composition of the present invention can alleviate the inflammatory response in the lungs by significantly reducing the release of inflammatory factors in the lungs of acute lung injury, enhance the antioxidant capacity by increasing the content of antioxidant enzymes, and can also inhibit protein expression. Signal transduction; and there are no clinical adverse reactions and toxic side effects in the whole course of treatment, and it has a significant clinical effect. The above results confirm that the pharmaceutical composition of the present invention has a good therapeutic effect on acute lung injury.

Claims

Application of a pharmaceutical composition in the preparation of a medicament for the treatment of acute lung injury, wherein the active ingredient of the pharmaceutical composition is made of the following raw materials in parts by weight:

Ephedra 52-86; Gypsum 194-324; Forsythia 194-324; Scutellaria 78-130; Mulberry Bark 194-324; Bitter Almond 78-130; Qianhu 78-130; Pinellia 78-130; Tangerine Peel 78-130 Fritillaria 78-130; Arctium lappa 78-130; Honeysuckle 78-130; Rhubarb 39-65; Platycodon 46-76; Licorice 39-65.
The application according to claim 1, wherein the active ingredient of the pharmaceutical composition is made of the following raw materials in parts by weight:

Ephedra 52; Gypsum 324; Forsythia 194; Scutellaria 78; Mulberry 194; Bitter Almond 130; Qianhu 78; Pinellia 130; Tangerine Peel 78; Fritillaria 78; Burdock Seed 130; Honeysuckle 130; Rhubarb 39; Platycodon 76; Licorice 65.
The application according to claim 1, wherein the active ingredient of the pharmaceutical composition is made of the following raw materials in parts by weight:

Ephedra 86; Gypsum 194; Forsythia 324; Scutellaria 130; Mulberry Bark 324; Bitter Almond 78; Qianhu 130; Pinellia 78; Tangerine Peel 130; Fritillaria 130; Burdock 78; Honeysuckle 78; Rhubarb 65; Platycodon 46; Licorice 39.
The application according to claim 1, wherein the active ingredient of the pharmaceutical composition is made of the following raw materials in parts by weight:

Ephedra 69; Gypsum 259; Forsythia 259; Scutellaria baicalensis 104; Mulberry bark 259; Bitter almond 104; Qianhu 104; Pinellia 104; Licorice 52.
The application according to claim 1, wherein the active ingredient of the pharmaceutical composition is made of the following raw materials in parts by weight:

Ephedra 55; Gypsum 254; Forsythia 318; Scutellaria baicalensis 107; Mulberry bark 203; Bitter almond 107; Qianhu 82; Pinellia 105; Platycodon 60; Licorice 50.
The use according to any one of claims 1 to 5, wherein the bitter almond is fried bitter almond, the fritillary is fritillary fritillary, the honeysuckle is the Lonicera japonica, and the Pinellia ternata is the Qing Pinellia.
The use according to any one of claims 1-6, wherein the active ingredient of the pharmaceutical composition is prepared by a method comprising the following steps:

A. Take the mother-of-pearl and crush it;

B. Extract ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb with solvent and concentrate to obtain a concentrate;

C. Extract gypsum, mulberry bark, Peucedanum praeruptorum, tangerine peel, mountain silver flower, platycodon, and licorice with solvent and concentrate to obtain a concentrate;

D. Combine the concentrate of step B and the concentrate of step C, and add the powder obtained in step A to the concentrate to obtain the pharmaceutical composition of the present invention.
The use according to any one of claims 1-7, wherein the active ingredient of the pharmaceutical composition is made by a method comprising the following steps:

A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, bitter almonds, pinellia, burdock seeds, rhubarb and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 times the amount for the first time , Add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to heat the clear cream with a relative density of 1.14-1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, honeysuckle, platycodon, and licorice according to the ratio of the prescription, add water and decoct twice for 1-4 hours each time, add 9-11 times the amount for the first time, and then add 9-11 times the amount for the second Add 7-9 times the amount each time, combine the decoction, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure the clear ointment with a relative density of 1.14-1.16, combine with the clear ointment obtained in step B, and set aside;

D. The clear ointment obtained by combining the fine powder obtained in step A and the concentrate of step C together constitutes the active ingredient of the pharmaceutical composition.
The use according to any one of claims 1-8, wherein the drug is a tablet, capsule, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray or The form of injection.
The use according to claim 9, wherein the tablet is made by a method including the following steps:

A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, bitter almonds, pinellia, burdock seeds, rhubarb and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 times the amount for the first time , Add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to heat the clear cream with a relative density of 1.14-1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, honeysuckle, platycodon, and licorice according to the ratio of the prescription, add water and decoct twice for 1-4 hours each time, add 9-11 times the amount for the first time, and then add 9-11 times the amount for the second Add 7-9 times the amount each time, combine the decoction, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure the clear ointment with a relative density of 1.14-1.16, combine with the clear ointment obtained in step B, and set aside;

D. Spray dry the combined cleansing paste obtained in step C, and collect the spray powder for later use;

E. The spray powder obtained in step D and the fine powder obtained in step A are prepared by using ethanol as a binder to prepare a soft material, and then sieving to granulate; and it is obtained by pressing.
The use according to claim 10, wherein the tablet is made by a method including the following steps:

A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, bitter almonds, pinellia, burdock seeds, rhubarb, and 50% ethanol to extract 2 times, each 3 hours, 10 times the amount for the first time, and 6 times for the second time. Double the volume, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure a clear paste with a relative density of 1.15 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, honeysuckle, platycodon, and licorice according to the proportion of the prescription, add water and decoct twice for 2 hours each time, 10 times the amount for the first time and 7 times for the second time The amount, the decoction is combined, filtered, the filtrate is decompressed to recover ethanol, and the filtrate is concentrated to a clean ointment with a relative density of 1.15 at 60°C, combined with the clean ointment obtained in step B, and set aside;

D. Spray dry the combined cleansing paste obtained in step C, and collect the spray powder for later use;

E. The spray powder obtained in step D and the fine powder obtained in step A are prepared with ethanol as a binder to make a soft material, sieved, granulated and dried, and then sieved. Add sodium starch glycolate, microcrystalline cellulose, and magnesium stearate, and mix well , Compressed tablets are available.
The use according to any one of claims 1-11, wherein the acute lung injury is manifested as an increase in cytoinflammatory factors.
The use according to claim 12, wherein the cell inflammatory factor is selected from IL-6, TNF-α, NE and MCP-1.
The use according to any one of claims 1-13, wherein the acute lung injury is manifested by increased protein expression.
The use according to claim 14, wherein the protein is selected from NF-κB P65, IκB-α, p-IκB-α and IKKβ.