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

Abstract

The invention relates to the technical field of medicines, in particular to application of a Tibetan medicine composition in preparation of a medicine for preventing or treating acute lung injury. The traditional Chinese medicine composition has a good treatment effect on the LPS-induced acute lung injury of rats by matching the raphanus sativus, the bergenia purpurascens, the shellac and the liquorice according to a specific ratio.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of a Tibetan medicine composition in preparing a medicine for preventing or treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a common clinically critical illness with a high mortality rate, seriously threatening the lives of critically ill patients. ALI is defined as diffuse interstitial and alveolar edema caused by damage to pulmonary capillary endothelial cells and alveolar epithelial cells during non-cardiac illnesses such as severe infection, shock, trauma, and burns, leading to acute hypoxic respiratory insufficiency or respiratory failure.

[0003] Acute lung injury can be treated with medication, most commonly with bronchodilators such as salbutamol sulfate and corticosteroids such as hydrocortisone to reduce inflammation and relieve symptoms. However, these medications can cause adverse reactions such as insomnia, nausea, vomiting, dizziness, and bronchitis. Traditional Chinese medicine (TCM) offers minimal side effects and a holistic approach. Therefore, there is a continued need to develop new TCMs with improved efficacy and minimal toxicity. Summary of the Invention

[0004] Therefore, the present invention aims to provide a Tibetan medicine composition for use in preparing a medicament for preventing or treating acute lung injury. The medicament has the advantages of good therapeutic effect and low toxic and side effects on acute lung injury.

[0005] In terms of weight, the Tibetan medicine composition includes the following raw materials: 180-220 parts of Radish, 140-180 parts of Bergenia, 80-120 parts of Lithospermum officinale and 80-120 parts of Licorice.

[0006] Furthermore, the Tibetan medicine composition includes the following raw materials in parts by weight: 200 parts of Radish, 160 parts of Bergenia, 100 parts of Lithospermum officinale and 100 parts of Licorice.

[0007] Furthermore, the preparation method of the Tibetan medicine composition includes mixing Radish, Bergenia, Lithospermum officinale and Licorice and extracting them according to a conventional extraction method or extracting them separately according to a conventional extraction method and then mixing them.

[0008] Furthermore, the conventional extraction method includes one or more of decoction extraction, maceration extraction, reflux extraction, and ultrasonic extraction; and / or, the extraction solvent is selected from water; and / or, the number of extractions is at least 1; and / or, the extraction time is at least 30 minutes; and / or, the ratio of the mass of the extraction solvent to the weight of the raw material is ≥2.

[0009] Furthermore, the Tibetan medicine composition is added or not added with a pharmaceutically acceptable carrier and prepared into a pharmaceutical preparation according to a conventional preparation method;

[0010] Preferably, the pharmaceutical preparation is an extract, decoction, powder, granule, tablet or capsule;

[0011] Preferably, the pharmaceutically acceptable excipient is selected from at least one of a pharmaceutically acceptable solvent, a binder, a disintegrant, a filler, a lubricant, and a glidant.

[0012] The present invention also provides a method for preparing Tibetan medicine granules, which comprises the following steps:

[0013] Radish, Bergenia, Lithospermum officinale and Licorice are mixed, and an extraction solvent is added for decoction to obtain an extract; the extract is concentrated and dried, and with or without auxiliary materials, Tibetan medicine granules are prepared according to conventional processes.

[0014] Furthermore, during the decoction extraction process, the number of extractions is 1-4 times, and the time for each extraction is 30-120 minutes; the ratio of the mass of the extraction solvent to the weight of the raw material drug is 6-12.

[0015] Furthermore, the excipients include one or more of dextrin, maltodextrin, sucrose, microcrystalline cellulose, lactose, and mannitol; and / or the mass of the excipients accounts for 50-90% of the total mass of the Tibetan medicine granules.

[0016] Furthermore, based on the mass of the dextrin dry matter, the mass content of glucose is 5%-11%.

[0017] The present invention also provides Tibetan medicine granules prepared by any of the above-mentioned preparation methods of Tibetan medicine granules.

[0018] The present invention also provides a Tibetan medicine granule prepared by any of the above-mentioned methods, wherein the Tibetan medicine granule has at least one of the following uses:

[0019] A. Use in the preparation of a medicament for preventing or treating acute lung injury;

[0020] B. Use in the preparation of medicines with antitussive effects;

[0021] C. Use in the preparation of medicines with expectorant effect;

[0022] D. Use in the preparation of drugs with anti-inflammatory effects.

[0023] The technical solution of the present invention has the following advantages:

[0024] 1. The Tibetan medicine composition provided by the present invention has a good therapeutic effect on LPS-induced acute lung injury in rats by using radish, bergenia, lithospermum officinale and licorice in a specific ratio.

[0025] 2. The present invention provides a method for preparing Tibetan medicine granules, comprising the following steps: mixing Radish, Bergenia, Lithospermum officinale, and Licorice root, decocting and extracting with an extraction solvent to obtain an extract; concentrating and drying the extract, and then preparing the Tibetan medicine granules according to conventional processes with or without the addition of excipients. This method is simple, easy to operate, and suitable for industrial production. In particular, the use of dextrin containing 5% to 11% glucose significantly improves the granules' fluidity, formability, solubility, and other characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 is the HE-stained section of the lung of rats in the normal group (×200);

[0028] Figure 2 is the HE-stained section of the lung of rats in the model group (×200);

[0029] Figure 3 is the HE-stained section of the lung of rats in the positive drug group (×200);

[0030] Figure 4 is the HE-stained section of the lung of rats in the low-dose group (×200);

[0031] Figure 5 is the HE-stained section of the lung of rats in the medium-dose group (×200);

[0032] Figure 6 is the HE-stained section of the lung of rats in the high-dose group (×200);

[0033] Figure 7 The Tibetan medicine granules of the present invention affect the inflammatory factors TNF-α, IL-6 and IL-1β in lung tissue. Note: p<0.05 (x±s, n=6) compared with the model group.

[0034] Figure 8The Tibetan medicine granules of the present invention have an effect on the cough latency and the number of coughs at different time points in rats. Note: p<0.05 (x±s, n=6) when comparing different groups with the model group.

[0035] Figure 9 is the absorbance standard curve for the phenol red excretion experiment;

[0036] Figure 10 This is the effect of the Tibetan medicine granules of the present invention on the excretion of phenol red. Note: p<0.05 (x±s, n=6) compared with the model group. DETAILED DESCRIPTION

[0037] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0038] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0039] All medicinal materials have passed quality control and conform to the standards of the Pharmacopoeia of the People's Republic of China. Quantities are expressed by weight. Coarse crushed material refers to particles less than 1 cm in length at the largest point and all particles larger than the size of a No. 1 sieve.

[0040] Example 1

[0041] This embodiment provides a Tibetan medicine composition, comprising the following raw materials: 40 g of Radish, 20 g of Lithospermum officinale, 32 g of Bergenia, and 20 g of Licorice.

[0042] This embodiment also provides an extract of the above Tibetan medicine composition, which is prepared as follows:

[0043] Weigh the coarse crushed materials of four medicinal materials, namely, Radish, Lithospermum officinale, Bergenia chinensis, and Licorice, mix them, add water and boil them for extraction, boil them once, add water in an amount that is 6 times the weight of the medicinal materials, boil them for 60 minutes, collect the decoction, and rotary evaporate it under vacuum degree of -0.07 to -0.08 MPa and temperature of 50°C to a volume of about 200-400 ml to obtain an extract.

[0044] Example 2 Powder

[0045] This embodiment provides a Tibetan medicine composition, comprising the following raw materials: 40 g of Radish, 20 g of Lithospermum officinale, 32 g of Bergenia, and 20 g of Licorice.

[0046] This embodiment also provides a powder of the Tibetan medicine composition, which is prepared as follows:

[0047] Weigh the coarse crushed materials of four medicinal materials, namely, Radish, Lithospermum officinale, Bergenia chinensis, and Licorice, add water and boil them for extraction. Boil them for a total of 3 times, adding water 10 times the weight of the medicinal materials each time, and boil them for 90 minutes each time. Combine the decoctions, and rotary evaporate them under vacuum of -0.07 to -0.08 MPa and temperature of 50-55°C to a volume of about 200-400 ml to obtain an extract, which is spray-dried into a fine powder to prepare a dry extract powder, i.e., a powder.

[0048] Example 3 Particles

[0049] This embodiment provides a Tibetan medicine composition, comprising the following raw materials: 40 g of Radish, 20 g of Lithospermum officinale, 32 g of Bergenia, and 20 g of Licorice.

[0050] This embodiment also provides a granule of the Tibetan medicine composition, which is prepared as follows:

[0051] Weigh the coarse ground materials of four medicinal materials, namely, Radish, Lithospermum officinale, Bergenia chinensis, and Licorice, add water and boil them for extraction. Boil them for a total of three times, adding water 10 times the weight of the medicinal materials each time. Boil them for 90 minutes each time. Combine the decoctions and evaporate them under vacuum of -0.07 to -0.08 MPa and temperature of 50-55°C to a volume of about 200-400 ml to obtain an extract. Spray dry the extract into 0.475 g of fine powder, add 1.525 g of dextrin and mix well, and dry granulate to prepare granules.

[0052] Example 4 Single Factor Experiment

[0053] 1. Water absorption test

[0054] Weigh 40 g of Radish, 20 g of Lithospermum officinale, 32 g of Bergenia, and 20 g of Licorice, soak them in water for 30 minutes, decoct them in 6 times the amount of water for 30 minutes, filter the liquid through 100 mesh, and weigh the mass of the wet pieces after decoction.

[0055] Decoction water absorption coefficient (K) = (mass of wet slices after decoction - mass of slices) / mass of slices, the mass of slices is 112 g.

[0056] After calculation, the water absorption coefficient is 1.77.

[0057] 2. Extraction method investigation test

[0058] Weigh 40g of Radish, 20g of Lithospermum officinale, 32g of Bergenia, and 20g of Licorice as one portion, for a total of 22 portions, and decoct them in water under the following conditions:

[0059] (1) The extraction time was 30 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively, and the number of extractions was 1; the weight of the extraction solvent was 6 times the weight of the medicinal material. Each condition was repeated twice, so the numbering based on (extraction time-parallel number) was 30-1, 30-2, 60-1, 60-2, 90-1, 90-2, 120-1, 120-2;

[0060] (2) The number of extractions was 1, 2, 3, and 4 respectively; the extraction time was 60 minutes; the weight of the extraction solvent was 6 times the weight of the medicinal material. Each condition was repeated twice, so the numbering based on (number of extractions - parallel number) was 1-1, 1-2, 2-1, 2-2, 3-1, 3-2, 4-1, and 4-2;

[0061] (3) The weight of water added was 6, 8, 10, and 12 times the weight of the medicinal material, respectively; the extraction time was 60 minutes; and the number of extractions was 1. Each condition was repeated twice, so the numbering based on (extraction multiple - parallel number) was 6-1, 6-2, 8-1, 8-2, 10-1, 10-2, 12-1, and 12-2;

[0062] Since the extraction conditions for 6-1, 6-2, 1-1, 1-2, 60-1, and 60-2 are all that the weight of the extraction solvent is 6 times the weight of the medicinal material, the number of extractions is 1, and the decoction time is 1 hour, only 60-1 and 60-2 are actually extracted, and other data can be quoted from these two sets of data.

[0063] Each sample extract obtained after decoction extraction was rotary evaporated to a volume of about 200-400 ml under vacuum conditions of -0.07 to -0.08 MPa and a temperature of 50°C to obtain an extract, which was weighed to obtain the total weight of the extract, recorded as m1. The extract was dried with reference to the method of "2201 Determination of Extracts: Determination of Water-Soluble Extracts" in the general rules of the 2020 edition (Part IV) of the "Chinese Pharmacopoeia". Specifically: Accurately weigh 25 g of extract (feeding amount, recorded as m) and place it in an evaporating dish that has been dried to a constant weight. After evaporating to dryness in a water bath, dry it at 105°C for 3 hours, cool it in a desiccator for 30 minutes, and obtain a dry extract. Quickly and accurately weigh the weight of the dry extract, recorded as m2. Two parallel portions.

[0064] The dry extract yield was calculated according to the following formula:

[0065]

[0066] m: feeding amount, unit: g;

[0067] m0: total mass of the original medicinal material, unit: g;

[0068] m1: total weight of extract, unit: g;

[0069] m2: weight of dry extract, unit: g.

[0070] The bergenin content in the dry paste was determined according to the bergenin content determination method under "Bergen" in Volume 1 of the Chinese Pharmacopoeia.

[0071] The results are shown in Table 1.

[0072] Table 1 Extraction results

[0073]

[0074]

[0075] From the above results, it can be seen that in the investigation of the number of extractions, with the increase in the number of extractions, the dry paste rate and bergenin content changed rapidly from 1 to 3 times, and changed slowly from 3 to 4 times. In addition, considering the actual large-scale production line in the later stage, the cost of 4 extractions was high and the benefits were slightly lower, so 1, 2, and 3 extractions were included in the orthogonal test. In the investigation of extraction time, with the increase in time, the dry paste rate and bergenin content changed rapidly from 30 to 90 minutes, and changed slowly from 90 to 120 minutes. In addition, considering the cost of actual large-scale production in the later stage, 30, 60, and 90 minutes were included in the orthogonal test. In the investigation of extraction multiples, with the increase in solvent multiples, the dry paste rate and bergenin content changed rapidly from 6 to 10 times, and changed slowly from 10 to 12 times. In addition, considering the cost of actual large-scale production in the later stage, 6, 8, and 10 times were included in the orthogonal test.

[0076] Example 5 Orthogonal experiment

[0077] The main factors affecting the extraction process are extraction time, number of extractions, and the multiple of solvent addition. After single-factor experiments, three influence levels have been screened for each of the three factors, which are used as orthogonal experimental conditions, as shown in Table 2.

[0078] 18 samples were weighed (each: 40 g of Radish, 20 g of Lithospermum officinale, 32 g of Bergenia, and 20 g of Licorice), and extraction studies were performed according to the orthogonal test in Table 3. The extracts were combined separately, and each sample extract obtained after decoction extraction was rotary evaporated to a volume of about 200-400 ml under a vacuum degree of -0.07 to -0.08 MPa and a temperature of 50°C to obtain an extract. The dry extract yield and the bergenin content (mg / g) in the dry extract were determined according to the method of Example 4, and the total mass of bergenin was calculated according to the following formula: Total mass of bergenin (mg) = Bergenin content in the dry extract (mg / g) × total mass of the dry extract (g). The results of the orthogonal test are shown in Table 3.

[0079] Table 2 Factor levels

[0080]

[0081]

[0082] Table 3 Orthogonal experiment results

[0083]

[0084] From the above results analysis, it can be seen that the influence of the three factors in comprehensive evaluation is A>B>C, and A, B, and C are significantly different (P<0.05). Therefore, the optimal process is: A3B3C3, that is, each extraction time is 90 minutes, decocted 3 times, and 10 times the amount of water is added each time.

[0085] Example 6 Excipient Screening Test

[0086] 1. Screening of auxiliary materials

[0087] (1) Dextrin, maltodextrin, sucrose, microcrystalline cellulose, lactose, and mannitol were used as excipients. 0.475 g of the dry extract powder prepared in Example 2 was added to each excipient at a mass ratio of dry extract powder to granules of 0.475:2, and the mixture was uniformly mixed. An appropriate amount of water was added and mixed uniformly. Granules were formed using a rocking granulator. The appearance and shape of the granules prepared with different excipients were compared, and the excipient suitable for the prescription was preliminarily screened based on the experimental results. The results are shown in the table below.

[0088] Table 4 Investigation of granulation with different excipient types

[0089]

[0090] Results: According to the granulation conditions of different excipients, maltodextrin, sucrose and lactose had too high viscosity, which led to serious agglomeration when mixed with the materials for granulation. Therefore, dextrin and microcrystalline cellulose were preliminarily screened as the best excipients.

[0091] (2) Dextrin and microcrystalline cellulose were used as auxiliary materials, respectively. 0.475 g of the dry extract powder prepared in Example 2 was taken, and each auxiliary material was added according to a mass ratio of dry extract powder to granules of 0.475:2, and mixed evenly. An appropriate amount of water was added and mixed evenly to form granules. Granules prepared from different auxiliary materials were compared and analyzed based on properties, bulk density, angle of repose, dissolution rate, and one-time forming rate as indicators, and the best auxiliary material was selected based on the experimental results.

[0092] 1) Characteristics

[0093] Granule properties: Observe the granules for particle size, uniformity, hardness, presence of lumps, and uniform color. The optimal score (100 points) is uniform size, moderate hardness, absence of lumps, and uniform color. Granules prepared with different excipients are scored based on these observations.

[0094] 2) Angle of repose

[0095] Use the fixed funnel method, connect the funnels in series and fix them at a height of 1 cm on the horizontal coordinate paper. Carefully pour the particles along the funnel wall into the top funnel until the tip of the particle cone formed on the coordinate paper touches the funnel mouth. The distance is H. Measure the radius R of the cone bottom from the coordinate paper and calculate the angle of repose α. The formula is: tg α =H / R.

[0096] 3) Melting rate

[0097] Accurately weigh about 5 g of particles and place them in a 250 mL conical flask. Accurately add 100 mL of water, stir and shake for 5 minutes, centrifuge at 3000 rpm for 15 minutes, discard the supernatant, and dry the residue at 80°C to constant weight. Accurately weigh the mass and calculate the solubility rate using the formula: solubility rate = (mass of dissolved particles / mass of particles) × 100%.

[0098] 4) Formability

[0099] The prepared granules were weighed and passed through a No. 1 sieve and then a No. 5 sieve. The granules that passed through the No. 1 sieve but not the No. 5 sieve were collected and weighed, and the molding rate was calculated using the formula: molding rate = (mass of granules after sieving / mass of granules before sieving) × 100%.

[0100] 5) Bulk density

[0101] Place the formed particles in a dry graduated cylinder, shake gently and determine the volume to be 5 mL, weigh the mass of the formed particles, and calculate the bulk density using the formula: ρ = m / V.

[0102] 6) Comprehensive score

[0103] Perform weight analysis on different assessment indicators and calculate the comprehensive score. The formula is:

[0104] Comprehensive score = forming rate × 20 / maximum forming rate + melting rate × 30 / maximum melting rate + property score × 20 / maximum score + minimum angle of repose × 15 / angle of repose + bulk density × 15 / maximum bulk density.

[0105] Table 5 Investigation of indicators of different types of excipients

[0106]

[0107]

[0108] Results: According to various indicators and comprehensive scoring results, dextrin was the most screened auxiliary material.

[0109] 2. Screening of excipient concentration

[0110] Dextrins with reducing sugar contents (calculated as glucose, dry basis) of 11%, 10%, and 5%, respectively, were used as auxiliary materials. 0.475 g of the dry extract powder prepared in Example 2 was taken and added to each auxiliary material at a mass ratio of dry extract powder to granules of 0.475:2, and mixed evenly. An appropriate amount of water was added and mixed evenly to form granules. Granules prepared with different auxiliary materials were compared and analyzed based on properties, bulk density, angle of repose, solubility rate, and one-time forming rate as indicators, and the best auxiliary material was selected based on the experimental results.

[0111] Table 6 Investigation of dextrin granulation with different glucose contents

[0112]

[0113] Results: According to the indicators, the forming rate of dextrin with medium glucose content was higher than that of dextrin with low glucose content, while the angle of repose of dextrin with high glucose content was larger than that of dextrin with low glucose content. However, after comprehensive scoring, it was found that dextrin with low glucose content was better than dextrin with medium glucose content. Therefore, dextrin with low glucose content was determined to be the best excipient.

[0114] 3. Screening of dextrin dosage

[0115] The current dosage is 0.475g per dose. Therefore, we prepared 1g, 2g, 3g, and 4g granules based on a 0.475g dry extract, adding different amounts of excipients for screening. The preparation method is as follows: Each group of dry extract powders is mixed with the excipients, and then an appropriate amount of water is added. Mixing thoroughly to form granules. Granules made with different excipients are compared and analyzed based on properties, bulk density, angle of repose, solubility, and one-shot forming rate. The optimal excipient dosage is selected based on the experimental results.

[0116] Table 7 Investigation on granulation with different amounts of excipients

[0117]

[0118] When the single dose is 1g, the dry paste powder and dextrin are mixed and added with water, which makes it impossible to make granules due to excessive viscosity, and thus the relevant indicators cannot be measured. Therefore, the single dose of 1g is not included in the screening range.

[0119] According to the results of various indicators, 2g was the best single-time dosage screened out, and the ratio of single-time dosage was 0.475g of dry paste powder and 1.525g of dextrin.

[0120] After comprehensive scoring based on the properties, bulk density, angle of repose, solubility rate, and molding rate of the particles as indicators for excipient screening, the final excipient process was to use dextrin with a low glucose content as an excipient, adding 1.525g of the excipient to 0.475g of the dry paste powder, for a total of 2g, as a single dosage.

[0121] Example 7 Acute Lung Injury

[0122] 1. Instruments, animals, and test drugs

[0123] (1) Instruments

[0124] Epoch microplate reader (Bio-Tek, USA), TGL-16K desktop high-speed refrigerated centrifuge (Hunan Xiangyi Centrifuge Factory), Olympus BX51 microscope (Olympus Corporation, Japan), 4% tissue fixative (Solarbio Company), automatic dehydrator (Kedi Instrument Equipment Co., Ltd., Jinhua City, Zhejiang Province), KD-BMⅡ computer biological tissue embedding machine (Kedi Instrument Equipment Co., Ltd., Jinhua City, Zhejiang Province), KD-BL freezing table (Kedi Instrument Equipment Co., Ltd., Jinhua City, Zhejiang Province), microtome (MICROM), manual pipette (Eppendorf, Germany), vaporizer (Shenzhen Ruiwode Company), qRT-PCR instrument (Thermo Fisher Scientific, USA).

[0125] (2) Animals

[0126] 180–220 g SPF rats were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd., license number SCXK(Xiang)-2019-0004. Primers were purchased from Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd., catalog number ZY00007; 4% tissue cell fixative and sterile enzyme-free water were purchased from Beijing Solebold Technology Co., Ltd., catalog numbers P1110-500 ml and R1600-100 ml, respectively; SYBR and reverse transcription kits were purchased from Shanghai Tolo Biotechnology Co., Ltd., catalog numbers 22208-01 and 22107, respectively; and phenol red was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number C151430050.

[0127] (3) Medicines

[0128] Pentoxetine citrate tablets were purchased from Sinopharm Rongsheng Pharmaceutical Co., Ltd. (National Medicine Approval No. H41022458), dexamethasone tablets were purchased from Guangdong Nanguo Pharmaceutical Co., Ltd. (National Medicine Approval No. H44024618), and ambroxol hydrochloride dispersible tablets were purchased from Shanxi Qianyuan Pharmaceutical Group Co., Ltd. (National Medicine Approval No. H20060254).

[0129] 2. Experimental methods

[0130] (1) Experimental grouping and drug administration

[0131] Rats were randomly divided into six groups, each consisting of six rats. The groups and dosing schedules were as follows: The three doses of Tibetan medicine granules were administered by gavage once daily (50 mg / kg, 100 mg / kg, and 200 mg / kg, respectively, based on the weight of the Tibetan medicine granules). The positive drug group received dexamethasone dispersion in water once daily (5 mg / kg). The normal control group and the ALI model (LPS) group received the same volume of saline.

[0132] Each group was administered medication for 7 consecutive days. After the last dose, rats in the model group, Tibetan medicine granule-treated group, and positive drug group were anesthetized with intratracheal LPS (5.0 mg / kg) to establish an ALI rat model. Except for the normal group, rats in all other groups were anesthetized with 10% chloral hydrate solution (0.3 mL / 100 g body weight) intraperitoneally. The rats were placed in a supine position on a laboratory table, their heads and limbs immobilized, and routinely disinfected. A longitudinal incision of approximately 1 cm was made in the neck skin at the cricoid cartilage, and the subcutaneous tissue was bluntly dissected to expose the trachea. A 1 mL syringe was inserted into the trachea between the two tracheal cartilages. The syringe was then held flat at approximately 10 degrees, and LPS solution was injected from the trachea into the lungs. The rats were then upright, rotated, and rocked side to side to evenly distribute the LPS solution in the lungs. Finally, the incision was sutured, thus establishing the endotoxin-induced ALI rat model. Model establishment was considered successful when the rats showed increased nasal mucus secretion, increased respiratory rate, and pulmonary rales. Rats in the normal group were instilled with an equal amount of phosphate buffered saline (PBS) into the trachea under the same conditions as described above.

[0133] (2) Measurement method

[0134] All rats were randomly selected 24 hours after modeling surgery, anesthetized and weighed. The chest and neck skin was cut open to expose the thoracic cavity. After ligating the right mainstem bronchus, a catheter was inserted into the mainstem bronchus to the lower bifurcation and 2 mL of PBS was injected to lavage the left lung. This was repeated three times. The collected bronchoalveolar lavage fluid (BALF) was placed in a pre-cooled 4°C centrifuge and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and stored for later use. The right lower lobe tissue was then excised, fixed in 4% paraformaldehyde solution, and embedded in paraffin. 0.4 mm thick sections were sliced ​​and stained with hematoxylin and eosin (H&E) to assess pathological changes.

[0135] At the same time, right middle lobe tissue was harvested for proinflammatory cytokine analysis. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to measure interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels in lung tissue.

[0136] 2. Data processing

[0137] The data involved in this study were statistically processed using SPSS22.0. All data were expressed as mean ± standard deviation (x ± s), and the t-test method was used to compare the differences between the two groups.

[0138] 3. Experimental results

[0139] (1) HE staining results

[0140] See Figure 1-6 As shown in the figure, HE staining revealed that compared with the normal control group, the model group experienced thickening of the alveolar walls and a corresponding reduction in alveolar cavities due to infiltration of inflammatory cells and swelling of vascular endothelial cells. Furthermore, disruption of the vascular barrier may have led to erythrocyte extravasation. Both positive drug and Tibetan medicine granules alleviated the corresponding pathological features in the model groups, with the degree of relief increasing with increasing dose of Tibetan medicine granules.

[0141] (2) qRT-PCR detection of inflammatory factors

[0142] See Figure 7 As shown, the Tibetan medicine granules of the present invention can reduce the mRNA levels of these inflammatory factors, among which IL-1β produced a significant difference, and TNF-α and IL-6 had a downward trend, but without statistical significance.

[0143] Example 8 Experimental Study on Antitussive

[0144] 1. Experimental methods

[0145] Rats were randomly divided into 6 groups, 6 rats in each group. The specific grouping and dosing conditions are as follows: 3 doses of Tibetan medicine granules treatment group, low dose group, medium dose group and high dose group: Tibetan medicine granules were administered by gavage to prepare a water dispersion, based on the weight of Tibetan medicine granules, once a day, and the daily doses were 50 mg / kg, 100 mg / kg and 200 mg / kg respectively. Positive drug group: Pentotheline citrate was administered by gavage to prepare a dispersion prepared by adding water, once a day, at a dose of 100 mg / kg. -1 / d -1 ; Normal group and ALI model group (LPS): The same volume of normal saline was administered by gavage.

[0146] Drug administration was continued for 7 consecutive days. All mice were fasted for 24 hours before the last dose. One hour after the last dose, all mice except those in the control group were placed in a transparent glass cover and sprayed with concentrated ammonia solution at a constant pressure for 20 seconds. The mice were then immediately removed. The latency period from the onset of coughing and the number of coughs within 3 minutes were recorded. The control group mice were sprayed with an equal volume of normal saline at a constant pressure. After collecting cough-related data, the eyeballs were immediately removed for blood collection. The mice were then sacrificed by cervical dislocation, and lung tissue was excised on an ice table for later use. The experiment observed and analyzed the effects of Tibetan medicine granules on the latency period of coughing and the number of coughs in rats.

[0147] 2. Data processing

[0148] The data involved in this study were statistically processed using SPSS22.0. All data were expressed as mean ± standard deviation (x ± s), and the t-test method was used to compare the differences between the two groups.

[0149] 3. Experimental results

[0150] This study aimed to evaluate the effect of Tibetan medicine granules on the antitussive effect in rats. The quantitative results of the experimental data are as follows: Figure 8 As shown:

[0151] Cough latency: Compared with the model group, the groups that received the medium-dose and high-dose interventions of positive drugs and Tibetan medicine granules showed significant differences.

[0152] Number of coughs: This study used 3 minutes and 5 minutes as nodes to count the number of coughs in mice. Among the four drug-dosing groups, the medium-dose and high-dose groups of Tibetan medicine granules A showed significant differences compared with the model group, while there was no significant difference between the positive drug group and the low-dose Tibetan medicine granules group.

[0153] Example 9 Experimental Study on Resolving Phlegm

[0154] 1. Experimental methods

[0155] Rats were randomly divided into six groups, each consisting of six rats. The specific grouping and dosing schedules were as follows: The three doses of Tibetan medicine granules were administered by gavage once daily (low-dose, medium-dose, and high-dose groups). The doses of 50 mg / kg, 100 mg / kg, and 200 mg / kg, respectively, were administered by gavage once daily. The positive drug group was administered by gavage once daily (40 mg / kg / day) with a dispersion of ambroxol hydrochloride dispersible tablets added to water. The normal control group and the ALI model (LPS) group were administered with the same volume of normal saline.

[0156] The drug was administered for 7 consecutive days. All mice were fasted for 24 hours before the last administration. 30 minutes after the last administration, all mice except the normal group were intraperitoneally injected with 0.5% phenol red saline solution (20 mL / kg). 30 minutes later, the eyeballs were removed to collect blood. The mice were killed by cervical dislocation and lung tissue was removed on an ice table for later use. After blood and lung collection, the trachea was exposed, the surrounding tissue of the trachea was stripped off, and a section of the trachea from the thyroid cartilage to the bronchial branch was cut. The trachea was placed in a centrifuge tube filled with 2 mL of saline, vortexed for 5 minutes, and then ultrasonically washed for 20 minutes. 1 mL of 50 g / L NaHCO3 solution was added and centrifuged at 1500 r / min for 10 minutes. The supernatant was collected and the absorbance at 546 nm was measured. The absorbance was compared with the phenol red standard curve to calculate the phenol red excretion. The standard curve is prepared as follows: Accurately weigh 25.0 mg of phenol red, dissolve it in an appropriate amount of 5% sodium bicarbonate solution, and transfer it to a 250 mL volumetric flask. Add 5% sodium bicarbonate solution to the mark. Then, dilute the solution to 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 3.0 μg / mL phenol red solutions. Measure the absorbance at a wavelength of 546 nm using a spectrophotometer. Plot a standard curve with phenol red concentration (X) as the horizontal axis and absorbance (Y) as the vertical axis.

[0157] 2. Data processing

[0158] The data involved in this study were statistically processed using SPSS22.0. All data were expressed as mean ± standard deviation (x ± s), and the t-test method was used to compare the differences between the two groups.

[0159] 3. Experimental results

[0160] The experimental results are as follows Figure 9 , Table 8 and Figure 10 shown.

[0161] Compared with the model group, after the administration of the positive drug and the Tibetan medicine granules of the present invention, the positive drug group and the high-dose group of the Tibetan medicine granules of the present invention had a significant upward effect on the excretion of phenol red.

[0162] Table 8 Effect of Tibetan medicine granules of the present invention on phenol red discharge

[0163]

[0164] Note: Comparison between different groups and the model group p<0.05 (x±s, n=6)

[0165] Experimental Conclusion

[0166] (1) The results of pathological sections and qRT-PCR detection of inflammatory factors show that the Tibetan medicine granules of the present invention have preventive and therapeutic effects on LPS-induced acute lung injury in rats.

[0167] (2) By counting and analyzing the rats' cough latency and the number of coughs within a certain period of time, it can be seen that the Tibetan medicine granules of the present invention can effectively alleviate the rats' cough induced by concentrated ammonia water.

[0168] (3) The phenol red excretion experiment showed that the Tibetan medicine granules of the present invention have good expectorant effect.

[0169] In summary, according to the results of the acute lung injury experiment, the cough relieving experiment and the expectorant experiment, it can be seen that the Tibetan medicine granules of the present invention have anti-inflammatory, cough relieving and expectorant effects.

[0170] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a Tibetan medicine composition in preparing a medicament for preventing or treating acute lung injury, characterized in that: In terms of weight, the Tibetan medicine composition includes the following raw materials: 180-220 parts of Radish, 140-180 parts of Bergenia, 80-120 parts of Lithospermum officinale and 80-120 parts of Licorice.

2. The use according to claim 1, characterized in that Calculated by weight, the Tibetan medicine composition includes the following raw materials: 200 parts of Radish, 160 parts of Bergenia, 100 parts of Lithospermum officinale and 100 parts of Licorice.

3. The use according to claim 1 or 2, characterized in that The preparation method of the Tibetan medicine composition comprises the following steps: mixing radish, bergenia, lithospermum officinale and liquorice, extracting the mixture according to a conventional extraction method, and then mixing the mixture to obtain the Tibetan medicine composition.

4. The use according to claim 3, characterized in that The conventional extraction method includes one or more of decoction extraction, maceration extraction, reflux extraction, and ultrasonic extraction; and / or, the extraction solvent is selected from water; and / or, the number of extractions is at least 1; and / or, the extraction time is at least 30 minutes; and / or, the ratio of the mass of the extraction solvent to the weight of the raw material is ≥2.

5. The use according to any one of claims 1 to 4, characterized in that The Tibetan medicine composition is added with or without a pharmaceutically acceptable carrier and prepared into a pharmaceutical preparation according to a conventional preparation method; Preferably, the pharmaceutical preparation is an extract, decoction, powder, granule, tablet or capsule; Preferably, the pharmaceutically acceptable excipient is selected from at least one of a pharmaceutically acceptable solvent, a binder, a disintegrant, a filler, a lubricant, and a glidant.

6. A method for preparing Tibetan medicine granules, characterized in that: The preparation method of the Tibetan medicine granules comprises the following steps: Mix radish, bergenia, lithospermum officinale and liquorice, add extraction solvent for decoction and extraction to obtain an extract; concentrate and dry the extract, add or do not add auxiliary materials, and prepare Tibetan medicine granules according to conventional processes; Optionally, the excipients include one or more of dextrin, maltodextrin, sucrose, microcrystalline cellulose, lactose, and mannitol; Optionally, the mass of the excipients accounts for 50-90% of the total mass of the Tibetan medicine granules.

7. The method for preparing Tibetan medicine granules according to claim 6, characterized in that: During the decoction extraction process, the number of extractions is 1-4 times, and the extraction time for each time is 30-120 minutes; the ratio of the mass of the extraction solvent to the weight of the raw material drug is 6-12.

8. The method for preparing Tibetan medicine granules according to claim 6, characterized in that: Based on the mass of the dextrin dry matter, the mass content of glucose is 5%-11%.

9. Tibetan medicine granules prepared by the preparation method according to any one of claims 6 to 8.

10. The Tibetan medicine granules prepared by the preparation method according to any one of claims 6 to 8 have at least one of the following uses: A. Use in the preparation of a medicament for preventing or treating acute lung injury; B. Use in the preparation of medicines with antitussive effects; C. Use in the preparation of medicines with expectorant effect; D. Use in the preparation of drugs with anti-inflammatory effects.

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