CN112999292B

CN112999292B - Lung-clearing intestine-clearing soup and application thereof in preparing medicine for treating lung injury

Info

Publication number: CN112999292B
Application number: CN202110246137.5A
Authority: CN
Inventors: 张志敏; 任培华; 关伟杰; 王鹏; 李振球; 黄琦玢; 薛玲娜; 张军
Original assignee: First Affiliated Hospital of Guangzhou Medical University
Current assignee: First Affiliated Hospital of Guangzhou Medical University
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2022-03-08
Anticipated expiration: 2041-03-05
Also published as: CN112999292A

Abstract

The invention discloses a Lifei Qingchang soup, comprising the following raw materials in parts by weight: Bupleurum 8-12 parts, Atractylodes 8-12 parts, Scutellaria baicalensis 8-12 parts, Jiang Pinellia 8-12 parts, Gardenia 8-12 parts servings, 10-15 servings of Scutellaria officinalis, 8-12 servings of Chuanxiong, 8-12 servings of rhubarb, 8-12 servings of licorice, and appropriate amount of Glauber's salt. The invention also discloses the preparation method of the Lifei Qingchang Decoction and the application in preparing the medicine for treating lung injury. The invention has a therapeutic effect on LPS-induced acute lung injury, inhibits the release of inflammatory factors, promotes the release of anti-inflammatory factors, and reduces the pathological damage of lung tissue.

Description

Lung-clearing intestine-clearing soup and application thereof in preparing medicine for treating lung injury

Technical Field

The invention relates to the field of medicines. More specifically, the invention relates to a lung-clearing and intestine-clearing soup and application thereof in preparing a medicament for treating lung injury.

Background

Bronchiectasis is the abnormal dilation of the bronchial tree, most of which is secondary to respiratory tract infection and bronchial obstruction, which causes the damage of the bronchial tube wall and the formation of persistent dilatation and deformation of the lumen, and is a common chronic bronchiectasis disease. The typical symptoms of this disease are chronic cough with profuse purulent sputum and repeated hemoptysis. Meanwhile, bronchiectasis is also a difficult disease to treat, compared with other airway diseases such as chronic obstructive pulmonary disease, bronchial asthma and the like, the attention degree is far behind, and the thinking and scheme of whole course treatment are lacked.

The prototype of the prescription of the lung-clearing and intestine-clearing decoction is formed probably from 2013, the effect is very good after patients with bronchiectasis, chronic obstructive pulmonary disease, asthma and the like are treated in the period, then clinical research is carried out continuously, the pathogenesis of bronchiectasis is continuously understood, finally the prescription is formed in 2016, and then the lung-clearing and intestine-clearing decoction is used for treating the patients with bronchiectasis. Since there is no radical treatment or drug approved for treating patients with bronchiectasis internationally, research efforts to prevent acute exacerbation, alleviate clinical symptoms, improve quality of life, and arrest progress of the condition remain clinical priorities.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide lung-clearing and intestine-clearing soup, a preparation method thereof and application thereof in preparing a medicament for treating lung injury, wherein the soup has a therapeutic effect on acute lung injury induced by LPS (low-pressure lipoprotein lipase), inhibits the release of inflammatory factors, promotes the release of anti-inflammatory factors and relieves pathological injury of lung tissues.

To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a lung-regulating and bowel-clearing soup comprising: the following raw materials in parts by weight: 8-12 parts of radix bupleuri, 8-12 parts of rhizoma atractylodis, 8-12 parts of scutellaria baicalensis, 8-12 parts of rhizoma pinelliae preparata, 8-12 parts of gardenia, 10-15 parts of caulis spatholobi, 8-12 parts of ligusticum wallichii, 8-12 parts of rheum officinale, 8-12 parts of liquorice and a proper amount of mirabilite.

Preferably, the lung-regulating and intestine-clearing soup is prepared into a pharmaceutically allowable dosage form.

The preparation method of the lung-regulating intestine-clearing soup comprises the following steps:

weighing radix bupleuri, rhizoma atractylodis, radix scutellariae, ginger processed pinellia tuber, fructus gardeniae, caulis spatholobi, rhizoma ligustici wallichii, rheum officinale and liquorice according to the weight parts, adding 6-8 times of water for decocting for 2-3 times, 1 hour each time, filtering, combining liquid medicines, concentrating crude medicines with the amount of 1.0-2.0g/mL, adding the mirabilite with the weight parts, filtering and drying to obtain main medicine powder.

Preferably, the drying is carried out at 70-100 deg.C under reduced pressure, during which time water is added to maintain a thick paste state, and drying for 5 h.

Preferably, the drying is spray drying, and the air inlet temperature is 160-180 ℃.

Preferably, it also comprises adjuvants including pharmaceutically acceptable carriers and excipients which are pharmaceutically acceptable, non-toxic and inert to humans and animals.

The application of the lung-clearing and intestine-clearing decoction in preparing a medicament for treating lung injury.

Preferably, the lung injury is lung tissue injury caused by recurrent bronchodilation, chronic obstructive pulmonary disease, or asthma, which is clinically manifested mainly by recurrent cough, expectoration, shortness of breath, or hemoptysis.

Preferably, the lung-clearing and intestine-clearing soup inhibits the release of inflammatory factors IL-6, IL-1 beta and TNF-alpha.

Preferably, the lung-clearing and intestine-clearing soup promotes the release of anti-inflammatory factors IL-4 and IL-10.

The invention at least comprises the following beneficial effects:

the lung-regulating intestine-clearing soup has a treatment effect on acute lung injury induced by LPS at a dosage of 1.15g/kg or more, and the action mechanism of the lung-regulating intestine-clearing soup can be related to the inhibition of the release of serum, alveolar lavage fluid and lung tissue inflammatory factors such as IL-6, IL-1 beta and TNF-alpha, the promotion of the generation and release of anti-inflammatory factors IL-4 and IL-10, the reduction of lung tissue pathological injury, the improvement of lung function, the improvement of weight loss caused by LPS and the improvement of the survival rate of mice.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is the effect of lung-clearing decoction of the present invention on the weight of mice with acute lung injury induced by conventional dose of LPS;

FIG. 2 is a graph showing the effect of lung-regulating intestine-clearing decoction of the present invention on the conventional level of LPS-induced blood in mice at conventional dose;

FIG. 3 is a graph showing the effect of lung-clearing decoction of the present invention on the levels of IL-6, IL-1. beta., TNF-. alpha., IL-4 and IL-10 in the serum of mice induced by LPS at a conventional dose;

FIG. 4 is a graph showing the effect of lung-clearing decoction of the present invention on the levels of conventional dose of LPS-induced alveolar lavage fluid IL-6, IL-1 β, TNF- α, IL-4 and IL-10 in mice;

FIG. 5 is a graph showing the effect of lung-clearing decoction of the present invention on the levels of IL-6, IL-1. beta., TNF-. alpha., IL-4 and IL-10 induced in mouse lung tissue by LPS at a conventional dose;

FIG. 6 shows the effect of lung-clearing decoction of the present invention on the conventional dose of LPS-induced lung function index of mice;

FIG. 7 shows the effect of lung-clearing decoction of the present invention on conventional dose of LPS induced pathological changes in lung histology;

FIG. 8 shows the effect of lung-regulating intestine-clearing decoction on the weight and survival rate of mice induced by a lethal dose of LPS;

FIG. 9 shows a 70%, 80%, 90% wetting agent granulation of the present invention;

FIG. 10 is a granulated form of 70%, 80%, 90% wetting agent of the present invention;

FIG. 11 is a granulated form of 70%, 80%, 90% wetting agent of the present invention;

FIG. 12 shows the 90% wetting agent granulation dissolution profile of the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

The preparation method of the lung-clearing and intestine-clearing soup comprises the following steps:

(1) preparing materials: the following raw materials in parts by weight: 8-12 parts of radix bupleuri, 8-12 parts of rhizoma atractylodis, 8-12 parts of scutellaria baicalensis, 8-12 parts of rhizoma pinelliae preparata, 8-12 parts of gardenia, 10-15 parts of caulis spatholobi, 8-12 parts of ligusticum wallichii, 8-12 parts of rheum officinale, 8-12 parts of liquorice and a proper amount of mirabilite;

(2) preparation: weighing radix bupleuri, rhizoma atractylodis, radix scutellariae, ginger processed pinellia tuber, fructus gardeniae, caulis spatholobi, rhizoma ligustici wallichii, rheum officinale and liquorice according to the weight parts, adding 6-8 times of water for decocting for 2-3 times respectively for 1 hour each time, combining filtrates, concentrating until the crude drug amount is 1.0-2.0g/mL, adding mirabilite, filtering and drying to obtain main drug powder.

The pharmaceutical preparation contains the lung-clearing and intestine-clearing decoction with effective treatment amount, and the rest is pharmaceutically acceptable carriers and excipients which are nontoxic and inert to human and animals.

The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical preparation of the present invention is used in the form of a dose per unit body weight. The extract of the present invention can be administered to a patient in need of treatment by oral administration or injection. For oral administration, it can be made into granule, mixture, tablet, sustained release tablet, controlled release tablet, capsule, dripping pill, pellet, suspension, emulsion, powder or granule (nanometer preparation), oral liquid, etc.; for injection, the composition can be made into sterilized aqueous or oily solution, sterile powder for injection, liposome or emulsion.

For example, when the compound aerosol is prepared, besides the proper propellant, the proper additive is selected mainly according to the physicochemical properties of the medicine to prepare the aerosol of a certain type so as to meet the requirements of clinical medication. The aerosol excipient mainly comprises a latent solvent and a propellant, wherein the latent solvent is glycerol, propylene glycol, ethanol, water or an ethanol-water mixed system, and the propellant mainly comprises trichloromonofluoromethane (F11), dichlorodifluoromethane (F12), dichlorotetrafluoroethane (F114) or a mixture of any two of the trichloromonofluoromethane, the dichlorodifluoromethane and the propellant. In addition, the aerosol can be added with surfactant, cosolvent, suspending agent, antioxidant, etc. to optimize the aerosol prescription.

For example, when the spray is prepared, ethanol, glycerol, water, propylene glycol or a mixture thereof is selected as a solvent; the fatty acid sorbitan substance, the polysorbate substance, the polyoxyethylene fatty alcohol ether substance, the polyoxyethylene-polyoxypropylene copolymer, the polyethylene glycol substance, the cyclodextrin substance, the sodium dodecyl sulfate, the cetyl trimethyl ammonium bromide or the mixture thereof is taken as a solubilizer; sodium dodecyl sulfate, laurocapram, lauric acid, sodium laurate, lecithin, oleic acid, poloxamer, choline phosphate, etc. or a mixture thereof as absorption enhancer.

The pharmaceutically acceptable carrier or excipient of the pharmaceutical formulation is not limiting and is presented as a clear illustration in the specification.

A first part: research on the Process

1. Purpose of experiment

And determining the optimal conditions of the processes of decoction, concentration and drying of the lung-clearing and intestine-clearing decoction.

2. Laboratory instruments and materials

An Agilent high performance liquid chromatograph, a UV detector, an LC-20AT pump, an SIL-20A autosampler and a Labsolution chromatographic workstation; high-speed traditional Chinese medicine pulverizer (YF-1000A, Yongzhan pharmaceutical machinery, Inc. of Ruian city); a refrigerated ultracentrifuge (H1850R, hunan instrument laboratory instruments development ltd.), a precision electronic analytical balance (FA/JA series, shanghai liangping instruments and meters ltd.), an electric-heated constant-temperature water bath (HWS-24, shanghai-heng science instruments ltd.), and an electric-heated air-blast drying oven (model 101-1AB, taister instruments ltd, tianjin).

Rhizoma Atractylodis (batch: HX20F01, Shingsu of origin), rhizoma Pinelliae Preparada (batch: YPB0G0008, Sichuan of origin), Scutellariae radix (batch: HX20E01, Shanxi of origin), Gardebuae fructus (batch: HX20E01, Hunan of origin), Ligustici Chuanxiong (batch: HX20F01, Sichuan of origin), bupleuri radix (batch: HX20F01, Hebei of origin), Rhei rhizoma (batch: HX20G01, Sichuan of origin), caulis Spatholobi (batch: HX20E01, Guangdong of origin), Glycyrrhrizae radix (batch: HX20E01, Gansu of origin), all of which are available from Guangdong and Xiang pharmaceutical Co.

Geniposide reference (batch number: MUST-19102310, purity: 99.84%, institute of Chengdu biological research of Chinese academy of sciences); baicalin reference (batch number: MUST-20030408, purity: 98.58%, institute of Chengdu biological research of Chinese academy of sciences); acetonitrile (Merck) is chromatographically pure, water is ultrapure and methanol is analytically pure.

3. Experimental methods and results

3.1 chromatographic conditions

Through optimization, the octadecylsilane chemically bonded silica is determined to be used as a filler (the length of a Kromasil C18 column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m); acetonitrile is taken as a mobile phase A, 0.38% phosphoric acid aqueous solution is taken as a mobile phase B, and the elution gradient is shown in the table 1; the flow rate is 1 mL/min; the column temperature was 25 ℃; the detection wavelength is 238nm and 321 nm.

TABLE 1 mobile phase gradiometer

3.2 creation of Standard Curve

3.2.1 preparation of control solutions

Preparing a gardenoside and baicalin mixed reference solution: accurately weighing jasminoidin and baicalin reference substances 0.42mg and 4.11mg respectively, placing in 10mL volumetric flask, adding methanol to scale, and shaking.

3.2.2 drawing of Standard Curve

The

above control solutions

1, 5, 10, 15, 20 and 25. mu.L were precisely pipetted into a liquid chromatograph, and the peak areas were recorded.

TABLE 2 geniposide sample introduction quality and peak area measurement results (238nm)

Taking the sample injection mass of the geniposide as a horizontal coordinate and the peak area as a vertical coordinate to draw a standard curve, calculating a regression equation, and obtaining a result: y is 1476.3x +25.208, r is 0.9990, which shows that the gardenoside peak area has good linear relation with the mass in the range of 0.0419-1.0483 μ g.

TABLE 3 baicalin sample introduction quality and Peak area measurement results (238nm)

Taking the sample injection mass of the baicalin as a horizontal coordinate and the peak area as a vertical coordinate to draw a standard curve, calculating a regression equation, and obtaining a result: y is 1145.5x +5.3715, r is 1, which shows that the baicalin peak area has good linear relation with the quality in the range of 0.4052-10.1291 μ g.

3.3 determination of the content of the decoction pieces

3.3.1 preparation of test solutions

Preparing a gardenia test solution: weighing the powder (sieving with a sieve of four numbers) about 0.l g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, weighing, ultrasonically treating for 20min, cooling, weighing again, supplementing the lost weight with methanol, shaking, and filtering. Precisely measuring 10mL of the subsequent filtrate, placing the subsequent filtrate in a 25mL measuring flask, adding methanol to the scale, and shaking up to obtain the final product.

Preparing a scutellaria baicalensis test solution: taking about 0.3g of the powder in the product, precisely weighing, adding 40mL of 70% ethanol, heating and refluxing for 3 hours, cooling, filtering, placing the filtrate in a 100mL measuring flask, washing the container and the residue with a small amount of 70% ethanol in several times, filtering the washing solution in the same measuring flask, adding 70% ethanol to the scale, and shaking uniformly to obtain the product.

3.3.2 determination of the content of the decoction pieces

Precisely sucking 10 μ L of each sample solution, injecting into liquid chromatograph, and measuring.

TABLE 4 determination of crude drug pieces content

3.4 determination of the number of times of decoction

3.4.1 preparation of Water-decocted test solution

Through preliminary experiments, the liquid absorption amount of the medicinal materials is about 2 times. Weighing 95g of the components except mirabilite according to the proportion of the prescription, adding 8, 6 and 6 times of water respectively, decocting for 3 times, 1 hour each time, filtering the liquid medicine respectively, concentrating to about 450mL, centrifuging for 15min at the rotating speed of 8000r/min, placing the supernatant into a 500mL measuring flask, adding water to the scale, and refrigerating for later use.

3.4.2 determination of cream yield

Precisely measuring the liquid medicine (equivalent to 4.75g of decoction pieces), pouring into an evaporating dish which is dried to constant weight, evaporating to dryness in a water bath, transferring into an oven at 105 ℃ for drying for 5h, taking out, placing in a dryer for cooling for 0.5h after completing the operation, precisely measuring, drying for 1h, cooling, and weighing. The above operation is carried out until the error of 2 times of weighing is not more than 3 mg. And calculating the paste rate.

TABLE 5 determination of cream yield

Note: water decoction 1 time solution: 25mL of the first decoction

Water decoction 2 times of solution: 25mL of the No. 1 decoction and 25mL of the No. 2 decoction

Water-decocting 3 times of solution: 25mL of the first decoction solution, 25mL of the second decoction solution, and 25mL of the third decoction solution

3.4.3 measurement of Retention rates of jasminoidin and baicalin

Precisely weighing 3.4.2 items, decocting in water for 1 time, 2 times, and 3 times, decocting in water for 1mL, placing in 10, and 5mL volumetric flasks respectively, adding water to scale, shaking, sucking the medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

TABLE 6 geniposide Retention assay results

TABLE 7 baicalin Retention Rate assay results

The results show that the difference of the retention rate of each component is not large when the raw materials are decocted for 3 times and decocted for 2 times. Combining clinical use experience, considering production efficiency and cost, and determining the decoction frequency to be 2 times. On the basis, an orthogonal test is carried out, and the decoction process is optimized.

3.5 orthogonal Experimental design

3.5.1 design of factor level gauge

On the basis of determining the decoction times to be 2 times, taking the retention rate of the effective components as an index, and adopting an orthogonal test to test and screen factors of water addition amount (A, times), decoction time (B, h) and soaking time (C, min), wherein the decoction process conditions of the formula are preferred. The following factor level table was designed.

TABLE 8 factor level table

3.5.2 orthogonal Experimental arrangements

3.5.2.1 preparation of test solutions

The experiment was arranged using an L9(34) orthogonal test Table. Weighing 47.5g of the components except Natrii sulfas according to the prescription ratio, decocting for 2 times according to the set scheme, filtering, mixing filtrates, centrifuging at 8000r/min for 15min, placing the supernatant in 1000mL measuring flask, adding water to desired volume, and refrigerating.

3.5.2.2 determination of paste yield

Respectively precisely measuring 25mL (equivalent to 4.75g decoction pieces) of the decocted liquid medicine, putting the decoction pieces into an evaporation dish which is dried to constant weight, evaporating the decoction pieces in a water bath to dryness, drying the decoction pieces in a hot air oven at 120 ℃ to constant weight, cooling the dried decoction pieces in a drier for 30min, precisely weighing the decoction pieces, and calculating the paste rate.

3.5.2.3 determination of retention rate of geniposide and baicalin

Precisely measuring 2mL of the liquid medicine obtained in the orthogonal experiments 1-9, respectively placing in 10mL volumetric flasks, adding water to the scale mark, shaking, sucking the liquid medicine, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

3.5.3 results of orthogonal experiments

3.5.3.1 determination of cream yield

TABLE 9 determination of cream yield

3.5.3.2 measurement results of component retention ratio of index

TABLE 10 geniposide Retention assay results and orthogonal evaluation

TABLE 11 baicalin Retention assay results and orthogonal evaluation

As can be seen from tables 10 and 11, the addition of water affects jasminoidin and yellowThe extraction effect of scutellarin is slightly influenced by the soaking time and the decocting time. Regarding the extraction effect of the geniposide and the baicalin, the water adding amount is more than 3 and more than 2 and more than 1, and the level 2 is similar to the level 3, so that the water adding amount is determined to be 2(8, 6 times) in order to save cost and reduce subsequent concentration time; the decocting time level 2 > level 3 > level 1, and in terms of the paste yield,

levels

2, 3 are slightly higher than level 1, so that the decocting time is determined to be level 2(1 h); there was no significant difference between the soaking

time levels

1, 2, 3, and no soaking was determined in combination with clinical medication experience. In conclusion, the decoction process is determined as A₂B₂C₁Namely, 8 and 6 times of water are respectively added, and the decoction time is 1 hour.

3.6 validation test

Experimental conditions A determined according to the decocting Process₂B₂C₁3 batches of sample validation process experiments were performed.

Weighing 47.5g of decoction pieces according to the proportion of the prescription, decocting twice, adding 8 and 6 times of water respectively, decocting for 1h, filtering, combining filtrates, centrifuging for 15min at the rotating speed of 8000r/min, placing the supernatant into a 1000mL measuring flask, adding water to a constant volume to scale, and refrigerating for later use.

(1) Determination of paste yield

Precisely weighing decoction equivalent to 4.75g of medicinal materials respectively, placing in an evaporating dish dried to constant weight, evaporating in water bath, drying in a hot air oven at 120 deg.C to constant weight, cooling in a drier for 30min, precisely weighing, and calculating to obtain paste rate.

TABLE 12 determination of cream yield

(2) Determination of retention rate of geniposide and baicalin

Precisely measuring 2mL of the above medicinal liquid, placing into 10mL volumetric flasks, adding water to the scale, shaking, sucking the medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

TABLE 13 geniposide Retention assay results

TABLE 14 baicalin Retention Rate assay results

The results show that under the optimized conditions, the retention rates of the geniposide and the baicalin are respectively about 76% and 77%, the extraction effect is ideal, and the process is stable, reasonable and feasible.

3.7 investigation of the concentration Process

3.7.1 preparation of concentrated medicinal liquid

Collecting medicinal liquid with crude drug concentration of 0.08g/mL, and measuring index component content.

Collecting two parts (300mL) of the above medicinal liquid, concentrating under reduced pressure in a rotary evaporator, concentrating one part until crude drug amount is 1.0g/mL, placing the concentrated solution in a 50mL measuring flask, and adding water to scale. Concentrating one part to soft extract with slight fluidity, placing the concentrated solution in 50mL measuring flask, and adding water to scale.

3.7.2 determination of component Retention ratio of each index in concentration Process

Preparing and measuring a test solution before concentration:

precisely measuring 1mL of the above medicinal liquid before concentration, placing in 5mL volumetric flasks respectively, adding water to scale mark, shaking, sucking the medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

Preparing and measuring a concentrated test solution:

precisely measuring the two concentrated medicinal liquids to obtain 1mL, placing into 50mL volumetric flasks respectively, adding water to scale mark, shaking, sucking medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

TABLE 15 measurement results of component retention of each index of the concentration process

As can be seen from table 15, the retention rates of the index components jasminoidin and baicalin are higher in the concentrated medicinal solution 1 than in the concentrated medicinal solution 2, which indicates that the concentration mode adopted by the concentrated medicinal solution 1 can prevent the loss of the effective components.

3.8 investigation of drying Process

3.8.1 determination of retention rate of each index component in reduced pressure drying process

Taking 50mL of concentrated liquid medicine, steaming three parts of the concentrated liquid medicine in water bath respectively to obtain thick paste, putting one part of the thick paste into a 50mL measuring flask, and adding water to the scale. Drying the other two parts at 70 deg.C under reduced pressure and 100 deg.C under normal pressure, adding water, keeping the thick paste state, drying for 5 hr, transferring to 50mL measuring bottle, and adding water to scale.

Precisely measuring 1mL of the above three medicinal liquids, placing into 50mL volumetric flasks, adding water to scale, shaking, sucking the medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

TABLE 16 measurement results of component retention of each index of the vacuum drying process

As can be seen from Table 16, the index components jasminoidin and baicalin were substantially retained under reduced pressure drying conditions, indicating that the reduced pressure drying manner can prevent loss of the effective components.

3.8.2 inspection of spray drying Process

3.8.2.1 determination of cream yield

Precisely absorbing 10mL of concentrated liquid medicine, placing the concentrated liquid medicine in an evaporating dish dried to constant weight, evaporating the concentrated liquid medicine in a water bath to dryness, drying the concentrated liquid medicine in a hot air oven at 120 ℃ to constant weight, placing the dried concentrated liquid medicine in a dryer for cooling for 30min, precisely weighing the concentrated liquid medicine, and calculating the paste rate. (the cream yield of the concentrated liquid medicine is 21.5%)

3.8.2.2 preparation of spray-dried product

Precisely measuring 100mL of concentrated liquid medicine, spray drying (inlet air temperature is 160 ℃, outlet air temperature is 90 ℃, inlet liquid speed is 9mL/min), and collecting spray dried powder.

3.8.2.3 preparation and determination of test samples

Preparing and measuring a concentrated liquid medicine test sample solution:

precisely measuring 14mL of the concentrated liquid medicine, placing in a 100mL measuring flask, adding water to scale, and shaking to obtain the concentrated liquid medicine test solution.

Precisely measuring 2mL of the concentrated medicinal liquid sample solution, placing in 10mL volumetric flasks respectively, adding water to the scale mark, shaking, sucking medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

Preparing and measuring a spray-dried test solution:

taking about 3.8g of spray-dried powder, precisely weighing, placing in a 25mL measuring flask, adding water to the flask to approximate the scale, performing ultrasonic treatment for 20min after vortexing, adding water to the scale, and shaking up to obtain spray-dried sample solution.

Precisely measuring 1mL of the above spray-dried sample solution, placing in 50mL volumetric flasks, adding water to the scale mark, shaking, sucking the medicinal liquid, filtering with microporous membrane (0.22 μm), and measuring by HPLC. Calculating the retention rate of geniposide and baicalin.

TABLE 17 determination of component Retention for each index of spray drying Process

As can be seen from Table 17, the retention rates of the index components jasminoidin and baicalin under the spray drying condition are more than 95%, which indicates that the effective components can be basically retained by adopting the spray drying mode.

A second part: formulation design

1. The daily prescription is as follows:

5g of rhizoma atractylodis, 5g of ginger processed pinellia tuber, 5g of radix scutellariae, 5g of cape jasmine fruit, 5g of ligusticum wallichii, 5g of radix bupleuri, 5g of rheum officinale, 7.5g of caulis spatholobi, 5g of liquorice and 2.5g of mirabilite.

An orthogonal verification process:

except for mirabilite 2.5g, decoction pieces 47.5g per day, and the paste rate is 25.5% by high-speed centrifugation in the orthogonal verification process. Calculated according to the calculation, the extract powder is taken by 25.5 percent by 47.5+2.5 to 14.6g each day;

2. a small trial process:

pilot plant process 1:

except for mirabilite 0.25kg, the decoction pieces are added for 4.75kg, and the small test process obtains 974g of powder by the micro-filtration theory, and the paste rate is 20.5 percent. Based on the calculation, 20.5% 47.5+2.5 ═ 12.2g of extract powder is taken every day.

Based on the above, 12-15g of extract powder is taken every day, and 4-5g of extract powder is taken every time.

Discussion of forming process:

4g of Qingyuan small test extract powder and 4.8g of soluble starch, wherein 70%, 80% and 90% ethanol are respectively used as wetting agents for granulation, and as shown in figure 9, small lumps which are high in viscosity and not easy to disperse are easily generated in the granulation process of 70% and 80% wetting agents; the 90% wetting agent is granulated, the viscosity is larger, the small lumps which are not easy to disperse are fewer, and the granules are more; the solubility examination showed marked turbidity.

A pilot plant process 2:

4g of Qingyuan small test extract powder and 4.8g of dextrin, 70 percent, 80 percent and 90 percent of ethanol are respectively used as wetting agents for granulation, as shown in figure 10, small lumps which are high in viscosity and not easy to disperse are easily generated in the granulation process of 70 percent and 80 percent of wetting agents, and more fine powder is generated; the 90% wetting agent is granulated, the viscosity is larger, the small lumps which are not easy to disperse are fewer, and the granules are more; the solubility was checked to be turbid.

A pilot plant process 3:

4g of Qingyuan small test extract powder, 4.3g of dextrin and 0.5g of lactose, 70 percent, 80 percent and 90 percent of ethanol are respectively used as wetting agents for granulating, as shown in figure 11, small lumps which are high in viscosity and not easy to disperse are easily generated in the granulating process of 70 percent and 80 percent of wetting agents, and more fine powder is generated; the 90% wetting agent is granulated, the viscosity is larger, the small lumps which are not easy to disperse are fewer, and the granules are more; the solubility was checked to be turbid.

And (4) a small trial process:

4g of Qingyuan small test extract powder, 3g of dextrin and 1.8g of lactose, and the wetting agent for granulating particles is 90% ethanol, as shown in figure 12, the granules have larger viscosity, less small lumps which are not easy to disperse, more particles and slightly turbid solubility inspection.

The adjuvant composition is therefore preferably: 4-5g of small test extract powder, 3g of dextrin and 1.8g of lactose. Namely lactose 1.5: and 2.5 of dextrin. Can meet the requirements of smooth granulation and better dissolubility.

If the viscosity is still large, a small amount of soluble starch can be added according to the proportion of the auxiliary materials, and the soluble starch is selected from the following components in percentage by weight: lactose 1.5: and 2.5 of dextrin.

3. Formulation of the preparation

Example 1: 5g of extract powder and 0.8 time of auxiliary material dosage are adopted, and a preparation formula is designed:

185g of rhizoma atractylodis, 185g of ginger processed pinellia tuber, 185g of radix scutellariae, 185g of cape jasmine fruit, 185g of rhizoma ligustici wallichii, 185g of radix bupleuri, 185g of rheum officinale, 277.5g of suberect spatholobus stem, 185g of liquorice, 92.5g of mirabilite, 1850g of total decoction pieces,

adding steviosin 4.5g, and making into 1000 g.

The granule is taken 3 times a day, 9g per bag, and 27g per day is equivalent to 50g of crude drug.

Each bag contains 5g of extract powder, 1.5g of lactose and 2.5g of dextrin.

Example 2: 5g of extract powder and 1 time of auxiliary material dosage are adopted, and a preparation formula is designed:

165g of rhizoma atractylodis, 165g of ginger processed pinellia tuber, 165g of radix scutellariae, 165g of cape jasmine fruit, 165g of ligusticum wallichii, 165g of radix bupleuri, 165g of rheum officinale, 247.5g of caulis spatholobi, 165g of liquorice, 82.5g of mirabilite and 1650g of total decoction pieces

Adding steviosin 4.0g to make into 1000 g.

Each bag is 10g, 3 times daily, and 30g of the granule is taken daily and is equivalent to 50g of the crude drug.

Each bag contains 5g of extract powder, 1.875g of lactose and 3.125g of dextrin.

Example 3: 5g of extract powder and 1.4 times of auxiliary materials are used for each time, and a preparation formula is designed:

139g of rhizoma atractylodis, 139g of ginger processed pinellia, 139g of scutellaria baicalensis, 139g of cape jasmine fruit, 139g of ligusticum wallichii, 139g of radix bupleuri, 139g of rheum officinale, 208.5g of caulis spatholobi, 139g of liquorice, 69.5g of mirabilite and 1390g of total decoction pieces

Adding steviosin 3.4g to make 1000 g.

The granule is taken 3 times a day, 33g per bag is equivalent to 50g of crude drug.

Each bag contains 5g of extract powder, 2.625g of lactose and 4.375g of dextrin.

And a third part: functional test verification

1. Materials and methods

1.1 Experimental animals

210 male BALB/c mice, 6-8 weeks old, weighing 20-22g, supplied by Schlekstaka laboratory animals Co., Ltd, Hunan; production license number of experimental animal: SCXK (Xiang) 2019-. The product is raised in an independent aeration cage box (IVC) of a standard laboratory animal laboratory of Chinese medicine university in Guangxi, and meets the requirements of SPF (specific pathogen free) grade. Controlling the room temperature to be 20.0-25.0 ℃ by using an air conditioner, controlling the relative humidity to be 40.0-70.0%, and illuminating for 12h/12h with alternating light and shade (6:00 turning on a lamp-18: 00 turning off the lamp); the mice are raised in non-toxic, high pressure resistant, high temperature resistant and corrosion resistant IVC cages, and are disinfected and cleaned regularly for a single sex. Feeding SPF mice with compound feed, and freely ingesting and drinking purified water.

1.2 drugs, reagents and instruments

1.2.1 medicine

1.2.1.1 Lung-regulating and intestine-clearing decoction

Lung-regulating and intestine-clearing decoction dry extract powder, crude drug equivalent 0.302g (dry extract powder)/g (crude drug amount), and tawny powder.

Storage conditions and stability: and (5) sealing and storing at room temperature. When in use, 1.15g of dry paste powder of lung-regulating and intestine-clearing decoction is taken, and is added with physiological saline for injection to 10mL, and the mixture is fully dissolved to obtain low-dose liquid medicine of 0.115 g/mL; taking 2.3g of lung-clearing and intestine-clearing decoction powder, adding 10mL of normal saline for injection, and fully dissolving to obtain a medium-dose liquid medicine of 0.23 g/mL; taking 4.6g of dry paste powder of lung-clearing and intestine-clearing decoction, adding physiological saline for injection to 10mL, and fully dissolving to obtain high-dose liquid medicine of 0.46 g/mL. Prepared immediately after use.

1.2.1.2 dexamethasone

Dexamethasone sodium phosphate injection (190321) purchased from Henan Ruhong pharmaceutical Co., Ltd. The content is as follows: 5mg/mL, Specification: 1 mL/piece. And (4) keeping the mixture in dark and in a sealed manner, and the effective period is 24 months. When in use, 1 dexamethasone sodium phosphate injection is taken, and is added with physiological saline for injection to 10mL, and is fully dissolved to obtain dexamethasone liquid medicine of 0.5mg/mL, which is prepared immediately after use.

1.2.2 reagents

LPS (S1732), cloudy day; mouse interleukin 6(IL-6), IL-1 beta and tumor necrosis factor alpha (TNF-alpha) ELISA kits, Invitrogen, lot #225266-022, #224603-014 and #234889-001, respectively; sodium pentobarbital, Merck, germany, batch No.: 120210.

1.2.3 instruments

The conventional detector for Mindray blood, Shenzhen Merrill biomedical electronics, Inc., model number: BC-5000 vet; electronic balance, mettler-toledo instruments shanghai ltd, model: ME 204E; low temperature high speed centrifuge, Eppendorf corporation, model: 5425R; enzyme linked immunosorbent assay (ELISA), BioTek, USA, model: synerggyh 1; pathological image analyzer, OLYMPUS, model: BX-60; tissue grinder, jieling instruments manufactures tianjin ltd, model: TP-24; animal ventilator, signal conditioner (AniRes animal lung function analysis system), beijing beland bokovic technologies ltd, model: RES3050, RES 1050.

1.3 methods

1.3.1 conventional dose of LPS induced acute Lung injury in mice

1.3.1.1 dosage design and grouping

The lung-clearing and intestine-clearing decoction was set to be low, medium and high in 3 dose groups, with doses of 1.15, 2.30 and 4.60g/kg (indicated by L, M, H in FIG. 1, XFJZT in FIGS. 2 to 6, LPS + XFJZT-1.15, LPS + XFJZT-2.30 and LPS + XFJZT-4.60 in FIG. 7, respectively), dexamethasone (5.0mg/kg) (indicated by DEX in FIG. 1, DEX in FIGS. 2 to 6 and LPS + Dex-5.0 in FIG. 7), model control (indicated by MO in FIG. 1 and LPS in FIGS. 2 to 7) and blank control (indicated by CON in FIGS. 1 and 7).

1.3.1.2 Molding and administration

4mg/mL pentobarbital sodium solution is prepared, an anesthetized mouse is injected in the abdominal cavity according to the weight (weight multiplied by 0.014), the anesthetized mouse is fixed on an operating table in the supine position, an oral cavity is opened by a mouse mouth gag, the position of epiglottis is found by the illumination of an operating lamp, a microinjector is inserted into the trachea of the mouse through the epiglottis, the LPS solution is injected into the lung according to 2.0mL/kg (2mg/mL), and the blank control group is given with physiological saline for equal volume sterile injection according to the weight. After the molding is finished, except for single administration of a dexamethasone group (5.0mg/kg), 0.5mg/mL dexamethasone liquid medicine is administered according to 10mL/kg intraperitoneal injection (i.p), each dose group of the lung-clearing and intestine-clearing decoction is subjected to intragastric administration according to 10mL/kg, a model control group is subjected to intragastric administration according to body weight (i.g), and the same volume of physiological saline for injection is administered for 1 time in 0h, 24h, 48h and 72h, and the total time is 4 times. Dynamic changes in survival were recorded for each group of mice.

1.3.2 lethal dose of LPS induced acute Lung injury in mice

1.3.2.1 dose design and grouping

The lung-clearing and intestine-clearing decoction was prepared in low, medium and high 3 dose groups, with doses of 1.15, 2.30 and 4.60g/kg (L, M, H in FIG. 8), respectively, and dexamethasone (5.0mg/kg) (DEX in FIG. 8), model control (MO in FIG. 8) and blank control (CON in FIG. 8).

1.3.2.2 Molding and administration

4mg/mL pentobarbital sodium solution is prepared, an anesthetized mouse is injected in the abdominal cavity according to the weight (weight multiplied by 0.014), the mouse is fixed on an operating table in the supine position, an oral cavity is opened by a mouse mouth gag, the position of epiglottis is found by the illumination of an operating lamp, a microinjector is inserted into the trachea of the mouse through the epiglottis, the LPS solution is injected into the lung according to 2.0mL/kg (7.5mg/mL), and the blank control group is given physiological saline for equal volume sterile injection according to the weight. After the molding is finished, except for single administration of a dexamethasone group (5.0mg/kg), 0.5mg/mL dexamethasone liquid medicine is administered according to 10mL/kg intraperitoneal injection (i.p), each dose group of the lung-clearing and intestine-clearing decoction is subjected to intragastric administration according to 10mL/kg, a model control group is subjected to intragastric administration according to body weight (i.g), and the same volume of physiological saline for injection is administered for 1 time in 0h, 24h, 48h and 72h, and the total time is 4 times. Dynamic changes in survival were recorded for each group of mice.

1.4 specimen Collection and detection

1.4.1 body weight

The body weight was weighed 1 time per Day, and the start of dosing (Day0), Day1, Day2, Day3, Day4 body weights were recorded.

1.4.2 detection of blood indices

1.4.2.1 detection of blood general indices

96h after the first administration, drawing out the eye to collect Blood, quickly sucking 45 μ L of whole Blood and fully mixing with 5 μ L of EDTA-K2 as anticoagulant, and detecting the total number of White Blood cells (WBC, 10) in Blood by using a conventional Blood detector⁹/L), neutrophils (NEUT, 10)⁹/L), lymphocytes (LYMPH, 10)⁹L) level.

1.4.2.2 detection of serum inflammatory factors

Collecting whole blood, placing in EP tube, centrifuging at 4 deg.C and 3000rpm for 20min after standing at room temperature for 2h, separating serum, subpackaging at-80 deg.C for detecting IL-6, IL-1 beta, TNF-alpha, IL-4 and IL-10 inflammatory factor levels of serum, and performing specific operation strictly according to ELISA kit instructions.

1.4.3 detection of inflammatory factors in alveolar lavage fluid (BALF)

After blood collection is finished, selecting half of mice in each group in sequence, killing the mice, quickly opening the thoracic cavity and exposing the neck trachea, injecting 0.3mL of PBS solution into the trachea by using a 1mL injector, repeatedly pumping back and injecting for 3 times to obtain alveolar lavage fluid, repeating the method for 3 times, combining the alveolar lavage fluid for 3 times, placing the alveolar lavage fluid in a low-temperature refrigerated centrifuge, centrifuging for 10min at 1600rpm at 4 ℃, collecting supernatant, detecting the levels of inflammatory factors IL-6, IL-1 beta, TNF-alpha, IL-4 and IL-10 of the alveolar lavage fluid by adopting an ELISA method, and specifically operating according to the specification of the ELISA kit.

1.4.4 Lung sampling and index detection

The right lung lobes were isolated from each group of the remaining mice, fixed with paraformaldehyde, embedded, sectioned, stained with eosin-Hematoxylin (HE), and the pathological changes of lung tissue were observed under light.

Rinsing the rest lung tissue with ice-cold physiological saline, sucking surface water with filter paper, weighing, recording, homogenizing with tissue grinder, centrifuging at 4 deg.C and 10000rpm for 10min, collecting supernatant, packaging, and freezing.

1.4.5 Lung function test

RL, Re, Cydn, PEF and MVV values were measured jointly using an RES3050 animal lung function instrument and an aniRes2005 software analysis system.

1.4.6 survival rates of mice

The number of deaths and the dynamic change in survival were recorded and counted for each group over 7 days.

1.5 data statistics

The Graphpad Prism 7 is adopted, the index results are expressed by means of the mean +/-standard deviation (X +/-S), the mean comparison between two groups adopts t test, and the measurement data comparison between multiple groups adopts one-way ANOVA and two-by-two comparison (LSD method). P <0.05 is statistically significant for differences.

2. Results

2.1 weight comparison of groups of mice

Compared with the model control group, the weight of the conventional dose test group is higher than that of the model control group in weight average after administration of the conventional dose test group and the dexamethasone group except that the weight of the lung-clearing and intestine-clearing soup low dose group Day1 is lower than that of the model control group, and the weight of the conventional dose test group and the dexamethasone group is not significantly different (P is more than 0.05).

The results show that LPS can cause the weight loss of BALB/c mice, and dexamethasone and lung-clearing decoction have the function of antagonizing the weight loss caused by LPS, and the detailed effect of the lung-clearing decoction on the weight loss of LPS-induced acute lung injury of mice is shown in figure 1 (

n＝20)。

2.2 routine blood

Compared with a blank control group, the level of WBC and NEUT in blood of mice in the model control group is obviously increased (P <0.001) and the level of LYMPH is obviously reduced (P <0.01) in the conventional dose test group; compared with a model control group, the level of total blood White Blood Cells (WBC), neutrophil granulocytes (NEUT) and Lymphocytes (LYMPH) of the lung clearing decoction mice is increased, wherein the WBC, NEUT and LYMPH levels of the rest groups are increased remarkably (P <0.05 or P <0.01 or P <0.001) except the WBC level difference of the low dose group and the medium dose group (P > 0.05).

The results show that LPS can cause WBC and NEUT level to be increased and LYMPH level to be reduced in BALB/c mice blood, and dexamethasone and lung-clearing decoction play a role in increasing WBC, NEU and LYM level, and the detailed result is that the influence of the lung-clearing decoction on the conventional level of the mouse blood is shown in figure 2

ABC is respectively a blank control group, a model control group, a lung-clearing and intestine-clearing soup low, medium and high dose group and a dexamethasone group from left to right.

2.3 serum inflammatory factors

Compared with a blank control group, the serum IL-6, IL-1 beta and TNF-alpha of the mice in the model control group are obviously increased (P is less than 0.05); compared with the model control group, the serum IL-6, IL-1 beta and TNF-alpha levels of mice in each dose group of the dexamethasone group and the lung-clearing and intestine-clearing decoction are remarkably reduced (P is less than 0.05). Compared with a blank control group, the serum IL-4 and IL-10 levels of the mice in the model control group are obviously reduced (P <0.01 or P < 0.05); compared with a model control group, the serum IL-4 and IL-10 levels of mice in the dexamethasone group are remarkably increased (P <0.01 or P <0.05), and the serum IL-4 and IL-10 levels of mice in each dose group of lung-clearing and intestine-clearing decoction are both increased, wherein the serum IL-4 and IL-10 levels of the mice in each dose group of the lung-clearing and intestine-clearing decoction are both remarkable (P <0.01 or P <0.05 or P < 0.005).

The results show that LPS can cause the increase of the serum inflammatory factors IL-6, IL-1 beta and TNF-alpha of BALB/c mice and the reduction of the levels of the anti-inflammatory factors IL-4 and IL-10, while dexamethasone and lung-clearing decoction have the function of antagonizing the change of the inflammatory factors caused by LPS, and the detailed picture of the effect of the lung-clearing decoction on the serum IL-6, IL-1 beta, TNF-alpha, IL-4 and IL-10 of the mice is shown in figure 3

ABCDE is respectively a blank control group, a model control group, a lung-clearing and intestine-clearing soup low, medium and high dose group and a dexamethasone group from left to right.

2.4 alveolar lavage fluid (BALF) inflammatory factor

Compared with a blank control group, the pulmonary alveolar lavage fluid IL-6, IL-1 beta and TNF-alpha levels of mice in a model control group are obviously increased in a conventional dose test group (P is less than 0.005); compared with the model control group, the mouse alveolar lavage fluid IL-6, IL-1 beta and TNF-alpha levels of the dexamethasone group are all obviously reduced (P <0.05), and the mouse alveolar lavage fluid IL-6, IL-1 beta and TNF-alpha levels of each dose group of lung clearing soup are all obviously reduced (P < 0.01). The model control mice had significantly reduced levels of alveolar lavage IL-4 and IL-10 (P <0.005) compared to the blank control group: compared with a model control group, the mouse in the dexamethasone group has the advantages that the pulmonary alveolar lavage fluid IL-4 and IL-10 levels are increased, wherein the IL-4 has significance, and the L-4 and IL-10 levels of the mouse in each dose of lung clearing decoction are significantly increased (P <0.01 or P < 0.05).

The results show that LPS can cause the increase of the level of IL-6, IL-1 beta and TNF-alpha of alveolar lavage fluid of BALB/c mice and the reduction of the level of anti-inflammatory factors IL-4 and IL-10, while dexamethasone and lung-clearing decoction have the function of antagonizing the change of the inflammatory factors of alveolar lavage fluid caused by LPS, and the detailed chart shows that the influence of the lung-clearing decoction on the level of IL-6, IL-1 beta, TNF-alpha, IL-4 and IL-10 of alveolar lavage fluid of mice is shown in figure 4

2.5 inflammatory factors of Lung tissue (lung tissue)

In the conventional dose test group, compared with a blank control group, the levels of IL-6, IL-1 beta and TNF-alpha in lung tissues of mice in the model control group are all obviously increased (P <0.01 or P <0.05 or P < 0.005); compared with a model control group, the levels of IL-6, IL-1 beta and TNF-alpha in alveolar lavage fluid of a dexamethasone group are all obviously reduced (P <0.01 or P <0.005), the levels of IL-6 in lung tissues of a medium-dose group are all obviously increased (P <0.05 or P <0.01), and the levels of IL-6 in a high-dose group of lung lavage fluid are all obviously reduced (P < 0.005); compared with a model control group, the IL-1 beta level difference of the lung-clearing intestine-clearing decoction low dose group is not significant (P >0.05), and the IL-1 beta level of the rest dose groups is significantly reduced (P <0.05 or P < 0.01); compared with a model control group, the tumor necrosis factor alpha (TNF-alpha) level of each dose group of the lung-clearing and intestine-clearing decoction is obviously reduced (P < 0.005); compared with a blank control group, the lung tissue IL-4 and IL-10 levels of the mice in the model control group are obviously reduced (P < 0.005): compared with the model control group, the mouse in the dexamethasone group has obviously increased level of IL-4 and IL-10 in the bleb lavage fluid (P <0.05 or P <0.01), and the level of IL-4 and IL-10 in the lung tissue of each dose group of the lung-clearing and intestine-clearing decoction is obviously increased (P <0.05 or P <0.01 or P < 0.005).

The results show that LPS can cause the IL-1 beta and TNF-alpha level to rise in the lung tissue of BALB/c mice, while dexamethasone and lung-clearing decoction have the function of antagonizing the IL-6 level rise of alveolar lavage fluid caused by LPS, and the detailed results are shown in figure 5 that the lung-clearing decoction has the effect on the IL-6, IL-1 beta, TNF-alpha, IL-4 and IL-10 level in the lung tissue of mice

2.6 pulmonary function index

Compared with a blank control group, the RL and Re levels of mice in a model control group are obviously improved, and the levels of Cydn, PEF and MVV are obviously reduced in a conventional dose test group; compared with the model group, except that the RL is not significant in the lung-clearing and intestine-clearing decoction low-dose group, the RL and Re levels of the rest dose groups and the dexamethasone group are significantly reduced; compared with the model group, the Cydn level, the PEF level and the MVV level (P level) of each of the rest dose groups and the dexamethasone group are obviously increased except that the Cydn level of the lung-clearing and intestine-clearing soup low-dose group and the dexamethasone MVV level have no significance<0.05 or P<0.01 or P<0.005), in detail, see FIG. 6 for the effect of lung-regulating and intestine-clearing decoction on the lung function index of mice

RL and Re are respectively arranged from left to right in A, and Cydn, PEF and MVV are respectively arranged from left to right in B.

2.7 histological and pathological changes of Lung tissue

In the conventional dose test group, the lung tissue tracheal mucosa of the blank control group mouse is smooth, structures such as normal alveolus, capillaries and the like can be seen, the alveolus does not have exudation, congestion, edema or inflammatory infiltration, the alveolar wall is thin, the distribution is neat, and the boundary is clear; the lung tissue of the mouse in the model control group is obviously changed, the damage and the collapse of the pulmonary alveoli are obviously extruded, the boundary is not clear, diffuse pulmonary alveoli exudation can be seen, and the lung mesenchyme is swollen and thickened; the lung tissue injury of the dexamethasone group and the lung-clearing and intestine-clearing decoction of each dose group is obviously improved, the alveolar collapse is obviously improved, the sthenia change is obviously relieved, the boundary is clear, the extrusion and inflammatory changes are obviously relieved, and the detailed view is shown in fig. 7 of the influence of the lung-clearing and intestine-clearing decoction on the pathological change of the lung histology.

2.8 lethal dose LPS induced acute lung injury mice weight and survival rate detection

Lethal dose test group, model control group weight average is significantly reduced compared with blank group (P)<0.001), compared with a model control group, the weight average of the body weight of each other dose group and the dexamethasone group is increased compared with that of the model control group after administration, except that the weight of the lung-clearing intestine-clearing decoction low dose group Day1 is lower than that of the model group, and no significant difference (P) exists>0.05). After LPS modeling, mice begin to die, the mice in the model group die on the fourth day, the survival rates of the dexamethasone group and the lung-clearing and intestine-clearing decoction groups are higher than those of the model group, the survival rate of the dexamethasone group in 6 days is 73.3%, the survival rates of the lung-clearing and intestine-clearing decoction groups in low, medium and high doses are 53.3%, 66.7% and 46.7% respectively, and the results show that the dexamethasone and the lung-clearing and intestine-clearing decoction have treatment and protection effects on the lung injury of the mice induced by the LPS with lethal dose (see the influence of the lung-clearing and intestine-clearing decoction on the weight and survival rate of the mice in FIG. 8 ((the effect of the lung-clearing and intestine-clearing decoction on the weight and survival rate of the mice)

n＝15)。

3. Conclusion

The applicant is continuously researched in clinic, and the lung-regulating and intestine-clearing decoction has obvious curative effects on reducing the number of acute attacks of patients, reducing the number of hemoptysis, reducing the hospitalization rate of the patients, greatly relieving the anxiety degree of the patients and the like in early clinical work. Meanwhile, under the experimental condition, the lung-clearing and intestine-clearing decoction has a treatment effect on acute lung injury induced by LPS (lipopolysaccharide), and the action mechanism of the lung-clearing and intestine-clearing decoction is probably related to the inhibition of the release of serum, alveolar lavage fluid and lung tissue inflammatory factors such as IL-6, IL-1 beta and TNF-alpha, the promotion of the generation and release of anti-inflammatory factors IL-4 and IL-10, the alleviation of pathological injury of lung tissue and the improvement of lung function. In addition, the lung-clearing and intestine-clearing soup also has the effects of improving weight loss caused by LPS and increasing the survival rate of mice.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. The preparation method of the lung-clearing and intestine-clearing soup is characterized by comprising the following steps:

preparing materials: the following raw materials in parts by weight: 8-12 parts of radix bupleuri, 8-12 parts of rhizoma atractylodis, 8-12 parts of scutellaria baicalensis, 8-12 parts of rhizoma pinelliae preparata, 8-12 parts of gardenia, 10-15 parts of caulis spatholobi, 8-12 parts of ligusticum wallichii, 8-12 parts of rheum officinale, 8-12 parts of liquorice and a proper amount of mirabilite;

preparation: weighing radix bupleuri, rhizoma atractylodis, radix scutellariae, ginger processed pinellia tuber, fructus gardeniae, caulis spatholobi, rhizoma ligustici wallichii, rheum officinale and liquorice according to the weight parts, adding 6-8 times of water for decocting for 2-3 times respectively, decocting for 1 hour each time, filtering, combining liquid medicines, concentrating until the crude drug amount is 1.0-2.0g/mL, adding mirabilite, filtering and drying to obtain main drug powder.

2. The preparation method of the lung-regulating intestine-clearing soup according to claim 1, wherein the lung-regulating intestine-clearing soup is prepared into a pharmaceutically acceptable dosage form.

3. The preparation method of the lung-regulating intestine-clearing decoction according to claim 1, wherein the drying is performed at 70-100 ℃ under reduced pressure, during which water is added to maintain a thick paste state, and the drying is performed for 5 hours.

4. The method for preparing the lung-clearing and intestine-clearing soup as claimed in claim 1, wherein the drying is spray drying, and the inlet air temperature is 160-.

5. The method for preparing the lung-regulating intestine-clearing soup according to claim 1, further comprising excipients including pharmaceutically acceptable carriers and excipients which are non-toxic and inert to humans and animals.

6. Lung-regulating and intestine-clearing soup obtained by the preparation method according to any one of claims 1-5.

7. Use of the lung-regulating and intestine-clearing decoction according to claim 6 in the preparation of a medicament for treating lung injury.

8. The use according to claim 7, wherein the lung injury is lung tissue injury caused by recurrent bronchiectasis, chronic obstructive pulmonary disease, asthma, with recurrent cough, expectoration, shortness of breath or hemoptysis as the main clinical manifestations.

9. The use of claim 7, wherein the lung-refreshing decoction inhibits the release of inflammatory factors IL-6, IL-1 β, TNF- α.

10. The use of claim 7, wherein the lung-clearing and intestine-clearing decoction promotes the release of anti-inflammatory factors IL-4, IL-10.