Preparation method and application of Tibetan medicine Qipeng pill

Abstract

The invention discloses a preparation method and application of a Tibetan medicine Qipeng pill. The Qipeng pill comprises Tibetan medicine active components and microcrystalline fibers, the microcrystalline fibers account for 30% of the weight of pellets, and the method comprises the following steps: uniformly mixing the active components and the microcrystalline fibers to obtain a mixture, adding 75% ethanol to prepare a soft material, filling the soft material into an extruder, extruding the soft material into strip columns, cutting the strip columns in a rolling machine, rolling the strip columns into pellets with uniform sizes, drying and sieving to obtain the Qipeng pill. Wherein the active component is prepared from fine powder of seven raw materials including fructus chebulae (denucleated), radix aconiti lateralis preparata, radix aucklandiae, rhizoma acori calami, Muslim myrrh, calculus bovis factitius and artificial musk. The Qipeng pill prepared by the method is good in roundness, good in friability, high in yield and uniform in size. In addition, the Qipeng pill also has the prospect of treating acute lung injury.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a preparation method and application of a Tibetan medicine Qipeng Pill. Background Art

[0002] Qipeng Pills are a Chinese medicine composition with antipyretic, analgesic, and anti-inflammatory effects. Chinese patent CN118697815A discloses a Chinese medicine composition, Qipeng Pills, made from seven ingredients: Terminalia chebula (de-cored), chub magnolia bark (processed), costus root, calamus tibetana, myrrha mukul, artificial bezoar, and artificial musk. The seven ingredients in this Chinese medicine composition, or Qipeng Pills, have the effects of clearing away heat and toxic substances, dispelling wind and dampness, reducing swelling and relieving pain, and killing parasites and treating pestilence. Clinically, it has anti-inflammatory, analgesic, and wind-relieving effects and is used to treat tonsillitis, pharyngitis, influenza, anthrax, rheumatic arthritis, neuralgia, stomachache, and toothache.

[0003] The weight composition of the raw materials of Qipeng Pills is as follows: 400-450 parts of Terminalia chebula (coreless), 50-60 parts of iron hammer (processed), 60-70 parts of costus root, 350-400 parts of Tibetan calamus, 25-35 parts of myrrh, 5-10 parts of artificial bezoar, and 5-10 parts of artificial musk.

[0004] CN118697815A discloses Qipeng Pills and a preparation method thereof. The steps are as follows: crush Terminalia chebula (core removed), iron hammer (processed), costus root, calamus tibetana, and myrrh into fine powder and sieve; the above seven ingredients, except artificial musk and artificial bezoar, are crushed into fine powder and sieved; the fine powder of artificial musk and artificial bezoar is added; the mixture is mixed and then an appropriate amount of water is added to make the pills.

[0005] During the research of Qipeng Pills, the inventors found that the existing pan-pill technology for preparing micropills had shortcomings, such as uneven micropills, insufficient roundness, and easy cracking. Therefore, the inventors specifically studied the preparation process of micropills and completed the present invention.

[0006] In addition, during the development of Qipeng Pills, it was discovered that Qipeng Pills also have therapeutic uses in treating acute lung injury. Summary of the Invention

[0007] The invention aims to provide a preparation method and application of Tibetan medicine Qipeng pills. The Qipeng pills prepared by the method have high roundness and good brittleness.

[0008] To achieve the purpose of the present invention, the following embodiments are provided.

[0009] In one embodiment, the present invention provides a method for preparing the Tibetan medicine Qipeng Pills, comprising an active ingredient and microcrystalline fiber, wherein the microcrystalline fiber accounts for 30% of the weight of the Qipeng Pills, wherein the active ingredient is prepared from fine powders of seven raw materials: Terminalia chebula (de-cored), ferox ferox (processed), costus root, calamus tatarinowii, myrrh, artificial bezoar, and artificial musk, and the method comprises the following steps:

[0010] 1) uniformly mixing the powder of the active component and the microcrystalline fiber to obtain a mixture;

[0011] 2) Add 75% ethanol as a wetting agent to the mixture to make a soft material;

[0012] 3) The soft material is fed into an extruder and extruded into a columnar shape, which is then cut into pellets or small pellets of uniform size in a spheronizer;

[0013] 4) The obtained micropellets are dried in a fluidized granulation coating machine and sieved.

[0014] Preferably, in the above-mentioned preparation method of the present invention, in step 3), the strip-out hole diameter is 2 mm.

[0015] Preferably, in the above-mentioned preparation method of the present invention, in step 3), the spheronization speed of the spheronization machine is 600 rpm.

[0016] Preferably, in the above-mentioned preparation method of the present invention, in step 3), the spheronization time of the spheronizer is 3 minutes.

[0017] Preferably, in the preparation method of the present invention, in step 4), the drying process is performed at a material temperature not exceeding 80° C. and the pellets are dried until the moisture content is ≤8.5%.

[0018] Preferably, in the preparation method of the present invention, the active component is composed of 400-450 parts of Terminalia chebula (de-core), 50-60 parts of iron hammer (processed), 60-70 parts of costus root, 350-400 parts of Tibetan calamus, 25-35 parts of myrrh, 5-10 parts of artificial bezoar, and 5-10 parts of artificial musk powder.

[0019] Preferably, in the preparation method of the present invention, the active component is prepared from 439.7 g of Terminalia chebula (de-cored), 56.4 g of iron hammer (processed), 67.4 g of costus root, 391.9 g of calamus, 29.6 g of myrrh, 8 g of artificial bezoar, 7 g of artificial musk or fine powder of the raw materials thereof by weight.

[0020] More preferably, the Qipeng pills of the present invention weigh about 0.01 g per pill, and the representative ingredient of the product is β-asarone (C 12 H 16 The content of O3) shall not be less than 0.8 mg / g.

[0021] In another embodiment, Qipeng Pills are used in the manufacture of a medicament for treating acute lung injury.

[0022] Preferably, for the above-mentioned use, the oral preparation of Qipeng Pills is in the form of micropills, which are taken orally twice a day, 9-20 pills at a time, each pill weighing about 0.01 gram.

[0023] Preferably, the oral preparation of Qipeng Pills is prepared by the above-mentioned method. have to.

[0024] Technical effects:

[0025] The preparation method of the Qipeng pills of the present invention solves the technical problems of poor uniformity, poor roundness, cracks, and poor brittleness of micropills prepared by the pan-pill technology using water as a wetting agent in the prior art by optimizing process parameters such as the proportion of excipients in the Qipeng pills, 30% microcrystalline fiber, 600 rpm, and 75% ethanol (aqueous solution) as a wetting agent. The preparation method of the present invention prepares Qipeng pills with high yield, high roundness and good brittleness, and good uniformity of micropill size.

[0026] At the same time, the present invention also found that Qipeng Pills have active and potential therapeutic effects on acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A picture of the pellets from the batch of Example 2 investigating the effect of wetting agents on the molding of Qipeng pills;

[0028] Figure 2 Effect of the amount of excipients used in Example 2 on the formation of Qipeng pills (pills from batches);

[0029] Figure 3 This is a diagram showing the inhibitory effect of Qipeng Pills on inflammatory factors in mice with LPS-induced acute lung injury;

[0030] Figure 4 The figure shows the pathological and qPCR results of Qipeng Pills against LPS-induced acute lung injury in mice. DETAILED DESCRIPTION

[0031] The following examples are provided to further illustrate the essence of the present invention, but are not intended to limit the scope of the present invention.

[0032] Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the contents of the present invention are still within the scope of protection of the present invention.

[0033] The following examples use an extrusion spheronization method to study the molding process of Qipeng Pills (QPW). Single-factor experiments were conducted to investigate the effects of wetting agent, excipient type, and excipient dosage on QPW molding. Based on the single-factor experiments, orthogonal experiments were performed to optimize the formulation and preparation process using the roundness, friability, and yield of the Qipeng Pills as indicators. The optimal preparation process was then verified, providing a basis for industrial production of the product. The active ingredient of the Qipeng Pills used in the examples was derived from the powder of Example 4 in Chinese Patent CN118697815A, which is incorporated by reference in its entirety.

[0034] Example 1 Preparation Technology of Qipeng Pills

[0035] Preparation of the active ingredient powder of Qipeng Pills

[0036] Recipe: Terminalia chebula (coreless) 439.7g, iron hammer (processed) 56.4g, costus root 67.4g, Tibetan calamus 391.9g, myrrh mukul 29.6g, artificial bezoar 8g, artificial musk 7g.

[0037] Preparation method: The above seven ingredients, except artificial musk and artificial bezoar, are ground into fine powder, sieved, and artificial musk and artificial bezoar powder are added and mixed to obtain the Qipeng Pills raw powder (powder). The raw powder can be amplified according to the weight and specific gravity of the formula to prepare the Qipeng Pills (micropills) of the present invention and the process research thereof.

[0038] Preparation process of Qipeng Pills:

[0039] Dry mixing: Add the weighed Qipeng Pills active ingredient powder and microcrystalline cellulose (weight ratio of 7:3) into the wet mixing granulator, set the stirring speed to 4-6 rpm and the chopping speed to 35-45 rpm, and let the materials dry mix for 5 minutes.

[0040] Preparation of soft material: spray 75% ethanol into the mixing granulator while stirring, set the stirring speed to 4-6 rpm, the chopping speed to 35-45 rpm, and stir at high speed to prepare the soft material.

[0041] Extrusion and spheronization: Select a strip aperture of 2mm, put the soft material into the extruder, set the extrusion speed to 20-30rpm, extrude into strips and columns, and then cut in a spheronizer with a spheronization speed of 200-600rpm to roll into micro-pellets of uniform size.

[0042] Drying: Add the prepared micro-pellets into the fluidized bed material cart for drying, adjust the inlet air temperature (≤120℃) so that the material temperature does not exceed 80℃, and dry until the moisture content of the micro-pellets is ≤8.5%. Discharge the material, place it in a clean plastic barrel, seal it, weigh it, and affix a material label.

[0043] Screening: Select the upper screen with 7 mesh and the lower screen with 9 mesh; screen the pellets, collect the qualified pellets, weigh them, and the obtained pellets can be coated according to conventional methods as needed.

[0044] Example 2 Investigation of influencing factors

[0045] The following single factor study examined the effect of

[0046] 2.1 Investigation of the influence of wetting agents

[0047] Referring to the process of Example 1, different proportions of ethanol aqueous solution or water were used as wetting agents in the soft material of Qipeng Pills. The spheronization speed was 600 rpm and the spheronization time was 3 min to prepare micropills. The pelleting effects of different wetting agents were investigated: water, 25% ethanol aqueous solution, 50% ethanol aqueous solution, 75% ethanol aqueous solution, and 95% ethanol. The results are shown in Table 1 and Figure 1 The results showed that it was impossible to produce round pellets of varying sizes by changing the ratio of water to ethanol, but a 75% ethanol aqueous solution produced a strip of soft material with the appropriate hardness. Therefore, based on the actual situation, a 75% ethanol aqueous solution was selected as the wetting agent.

[0048] The results showed that 75% ethanol was more suitable for preparing columnar soft materials as wetting agents, and relatively uniform spherical micropellets could be made after rounding. However, other concentrations of ethanol and water as wetting agents could not produce round micropellets or balls with uniform size.

[0049] Table 1. Results of investigation on the influence of wetting agent

[0050]

[0051]

[0052] 2.2 Investigation of the influence of excipients

[0053] Referring to the process of Example 1, the effect of the amount of excipients in the pellet soft material on the pellet forming and quality was investigated. The wetting agent was 75% ethanol aqueous solution, the spheronization speed was 600 rpm, and the spheronization time was 3 min. The effect of the amount of microcrystalline cellulose and magnesium stearate on the pelleting effect was investigated. The results are shown in Table 2 and Figure 2 The results showed that as the proportion of microcrystalline cellulose increased, the roundness value improved. Based on the actual situation, the proportion of microcrystalline cellulose in the micropellets was selected to be 30%. The lubricant magnesium stearate affected the quality of the micropellets, so it was ultimately decided not to add magnesium stearate.

[0054] Table 2. Excipient inspection results

[0055]

[0056]

[0057] 2.3 Effect of spheronization frequency on pellet formation

[0058] Referring to the process of Example 1, the effects of spheronization on pellet formation and quality were investigated. Based on the wetting agent and excipient investigations, a 75% ethanol-water solution was selected as the wetting agent, a 30% microcrystalline cellulose content was used, a spheronization time of 3 minutes, and spheronization frequencies of 400 rpm, 600 rpm, and 800 rpm were used to produce pellets. The results are shown in Table 3. The results show that as the spheronization frequency increases, the yield initially increases and then decreases, and the roundness improves. Friability initially decreases and then remains constant. Based on the actual situation, a spheronization frequency of 600 rpm was selected.

[0059] Table 3. Results of the investigation on rolling frequency

[0060] batch number Rolling frequency (rpm) Yield (%) Roundness Friability (%) 24082301 400 86.34 18.62° 0.45 24082302 600 90.26 16.32° 0.38 24082303 800 87.64 15.84° 0.39

[0061] 2.4 Rounding time inspection

[0062] Referring to the process of Example 1, the effect of spheronization time on pellet formation and quality was investigated. Based on the results of the wetting agent, excipient, and spheronization frequency investigations, a 75% ethanol-water solution was selected as the wetting agent, a 30% microcrystalline cellulose content, and a spheronization frequency of 600 rpm were selected. Spheronization times of 2, 3, and 4 minutes were designed, respectively, and pellets were produced according to the "3 Experimental Steps." The results are shown in Table 4. The results show that with increasing spheronization time, pellet yield initially increased and then decreased, and improved roundness and friability increased. Based on the actual situation, a spheronization time of 3 minutes was selected.

[0063] Table 4. Results of the rounding time investigation

[0064] batch number Spheronization time (rpm) Yield (%) Roundness Friability (%) 24122301 2 87.32 17.54° 0.34 24082302 3 89.23 16.46° 0.36 24082303 4 88.64 16.32° 0.40

[0065] 2.5 Orthogonal experimental design

[0066] Referring to the process of Example 1, based on the results of the single factor experiment, three different levels were set for the three key factors of microcrystalline cellulose dosage, spheronization frequency, and spheronization time, and the yield, roundness, and friability were used as the evaluation indicators for comprehensive scoring. An orthogonal test L9 (3 was performed using 3 factors and 3 levels. 4 ) to optimize the micropellet preparation process parameters to determine the optimal micropellet preparation process. Specific factors and level settings are shown in Table 5. Experiments were conducted using the corresponding parameters of the orthogonal test and the micropellet preparation process studied in the pilot experiment. The test results are shown in Table 6.

[0067] Table 5. Orthogonal test factor level table

[0068]

[0069] Table 6. Orthogonal test results

[0070]

[0071] *Note: Based on the importance of pellet evaluation, the weight coefficient of yield is set to 0.4, and the weight coefficients of roundness and friability are both set to 0.2.

[0072] Comprehensive score = (yield / maximum yield*0.4)*100+(minimum roundness / roundness*0.4)*100+(minimum friability / friability*0.2)*100

[0073] The results of the orthogonal experiment were analyzed visually and by variance analysis to determine the degree of influence of each factor on the preparation of Qipeng Pills and to screen and optimize the preparation process conditions. The variance analysis results are shown in Table 7.

[0074] Table 7. Analysis of variance

[0075]

[0076] Visual analysis and variance analysis revealed that the order of factors influencing pelleting performance was microcrystalline cellulose dosage > spheronization speed > spheronization time, and the optimal process combination was A2B2C2. A P value of less than 0.05 for microcrystalline cellulose dosage indicated that microcrystalline cellulose dosage significantly affected QWP pelleting performance, while spheronization frequency and spheronization time had no significant effect on the Qipeng Pills preparation process. Therefore, the optimal process was determined to be a 30% microcrystalline cellulose content, a spheronization speed of 600 rpm, and a spheronization time of 3 minutes.

[0077] 2.6 Validation of Qipeng Pills Preparation Process

[0078] Three batches of Qipeng Pills were prepared in parallel using the optimal preparation process from the orthogonal design. The yield, roundness, and friability of the pills were measured. The results are shown in Table 8. The results of the validation test were generally consistent with the orthogonal optimization results, demonstrating that the optimal preparation process conditions for the micropellet obtained from the orthogonal design were reasonable, feasible, and reproducible.

[0079] Table 8. Validation results of Qipeng pill preparation process

[0080] batch number Yield (%) Roundness Friability (%) Overall score 24122601 89.6 16.5 0.33 97.19 24122602 90.5 16.2 0.31 99.96 24122603 90.1 16.5 0.32 98.30

[0081] Example 3 Preparation of Qipeng Pills

[0082] Preparation of Qipeng Pills Active Combination Powder

[0083] Recipe: Terminalia chebula (coreless) 439.7g, iron hammer (processed) 56.4g, costus root 67.4g, Tibetan calamus 391.9g, myrrh mukul 29.6g, artificial bezoar 8g, artificial musk 7g.

[0084] Preparation method: The above seven ingredients, except artificial musk and artificial bezoar, are ground into fine powder, sieved, and artificial musk and artificial bezoar powder are added and mixed to obtain the Qipeng Pills raw powder (powder). The raw powder can be amplified according to the weight and specific gravity of the formula to prepare the Qipeng Pills micropills of the present invention and the process research thereof.

[0085] Preparation process of Qipeng Pills:

[0086] Dry mixing: Add the weighed active ingredient powder of Qipeng Pills and microcrystalline cellulose (7:3 by weight, accounting for 30%) into the wet mixing granulator, set the stirring speed to 4-6 rpm and the chopping speed to 35-45 rpm, and let the materials dry mix for 5 minutes.

[0087] Preparation of soft material: spray 75% ethanol into the granulator while stirring, set the stirring speed to 4-6 rpm, the chopping speed to 35-45 rpm, and stir at high speed to prepare the soft material.

[0088] Extrusion and spheronization: Select a strip aperture of 2mm, put the soft material into the extruder, set the extrusion speed to 20-30rpm, extrude into strips and columns, and then cut in a spheronizer with a spheronization speed of 600rpm and a spheronization time of 3 minutes to roll into micro-pellets of uniform size.

[0089] Drying: Add the prepared micro-pellets into the fluidized bed material cart for drying, adjust the inlet air temperature (≤120℃) so that the material temperature does not exceed 80℃, and dry until the moisture content of the micro-pellets is ≤8.5%. Discharge the material, place it in a clean plastic barrel, seal it, weigh it, and affix a material label.

[0090] Screening: Select the upper screen with 7 mesh and the lower screen with 9 mesh; screen the pellets, collect the qualified pellets, weigh them, and make pellets of uniform size.

[0091] Inspection: Moisture content, filling volume difference, and dispersibility time limit all comply with the relevant regulations for pills (General Principle 0108, Part IV, 2020 Edition of the Chinese Pharmacopoeia).

[0092] Detection of β-asarone content, the hallmark ingredient of Qipeng Pills:

[0093] Determined according to high performance liquid chromatography (General Rule 0512).

[0094] Chromatographic conditions: Octadecylsilane bonded silica gel as the filler; acetonitrile-0.1% phosphoric acid as the mobile phase; detection wavelength at 210 nm. The theoretical plate number calculated based on the β-asarone peak should be no less than 30,000.

[0095] Preparation of reference solution: Take an appropriate amount of β-asarone reference substance, accurately weigh it, dilute it with methanol to a reference substance mother solution containing 5 mg per mL, and gradually dilute the mother solution with methanol to prepare solutions with concentrations of 3.5584, 13.9, 27.8, 111.2, and 222.4 μg / mL.

[0096] Preparation of test solution: Take about 0.5 g of powder for preparing micropellets (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of methanol solution, weigh the weight, ultrasonicate for 10 minutes (power 250 W, frequency 50 kHz), let cool, weigh the weight again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0097] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0098] This product contains β-asarone (C 12 H 16 O3), not less than 0.8 mg / g.

[0099] Example 4: Effect of Qipeng Pills (Pellets) on Anti-Acute Lung Injury in Vivo

[0100] 1. Materials

[0101] experimental animals

[0102] C57bl / 6 mice were provided by Liaoning Changsheng Biotechnology Co., Ltd., male, 18-20 g, SPF grade.

[0103] 2. Experimental Methods

[0104] 2.1 Experimental groups and drug administration

[0105] After one week of adaptive feeding, C57B6L / J mice were randomly divided into: normal group (Control), model group (LPS), Qipeng Pills (micropellets) high-dose administration group, Qipeng Pills (micropellets) low-dose administration group, and dexamethasone positive drug group, with 10 mice in each group.

[0106] 2.2 Preparation of drug solution

[0107] Weigh 1g of CMC-Na and add 200ml of distilled water. Dissolve in a water bath and heat to obtain a 0.5% CMC-Na solution. Grind Qipeng Pills (pellets) and dissolve in a small amount of DMSO. Then resuspend them in PEG 400. Finally, add 0.5% CMC-Na in a ratio of DMSO:PEG400:0.5% CMC-Na (1:5:15). The positive drug dexamethasone (DEX) is directly dissolved in normal saline.

[0108] 2.3 Animal modeling and drug administration

[0109] LPS solution with a concentration of 5 mg / kg was prepared with normal saline and used daily. The drug-treated group and the positive drug group were gavaged with the drug every day for three days. The blank group and the model group were gavaged with the same volume of normal saline for three days. On the third day, LPS (20 μL / 20 g) was administered to the mice by nasal drops to create a model. The experimental groups were blank group, LPS group, drug doses of 240 mg / kg and 480 mg / kg, and dexamethasone group (5 mg / kg), with 10 mice in each group. The animals were killed 24 hours after modeling.

[0110] 2.4 Collection of mouse visceral tissues

[0111] The bodies of the killed mice were fixed on a foam board, and the heart, liver, spleen, lungs, and kidneys were removed by dissection. The surfaces were washed with physiological saline, and the surface moisture was absorbed with filter paper. The bodies were then fixed in a sufficient amount of 4% paraformaldehyde and stored at room temperature.

[0112] 2.5 H&E staining

[0113] Mouse lung tissue was immersed in 4% paraformaldehyde and fixed at room temperature for 24 hours before embedding and paraffin sectioning. The key steps for mouse tissue sectioning and staining are as follows:

[0114] (1) Dewaxing of paraffin sections to water: Place the sections in the order shown in Table 9.

[0115] Table 9. Paraffin section dewaxing procedure

[0116] Reagents time Environmentally friendly dewaxing liquid Ⅰ 20min Environmentally friendly dewaxing liquid Ⅱ 20min Anhydrous ethanol Ⅰ 5min Anhydrous ethanol Ⅱ 5min 75% alcohol 5min Distilled water wash -

[0117] (2) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0118] (3) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin solution for 5 min.

[0119] (4) Dehydration and sealing: Dehydrate the sections in the order shown in Table 10 and seal with neutral gum.

[0120] Table 10. Dehydration program

[0121] Reagents time Anhydrous ethanol I 5min Anhydrous ethanol II 5min Anhydrous ethanol Ⅲ 5min Xylene Ⅰ 5min Xylene Ⅱ 5min

[0122] (5) Microscopic examination, image acquisition and analysis.

[0123] 2.6 Immunohistochemistry

[0124] (1) Dewaxing of paraffin sections to water: Dewax the sections in the following order as shown in Table 9.

[0125] (2) Antigen retrieval: Microwave citric acid (pH 6.0) on medium heat for 8 minutes, then off for 8 minutes and then on medium-low heat for 7 minutes. During the retrieval process, prevent excessive evaporation of the buffer and do not allow the slides to dry out. After retrieval, allow the slides to cool naturally. Wash the slides three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each.

[0126] (3) Circle drawing and hydrogen peroxide blocking: Use a histochemical pen to draw a circle around the tissue, place the slices in 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes to block endogenous peroxidase, and place the slides in PBS (PH 7.4) and shake on a decolorization shaker to wash three times, each time for 5 minutes.

[0127] (4) Serum blocking: Drain PBS and add BSA (if the primary antibody is from goat, use 10% rabbit serum to block; if the primary antibody is from other sources, use 3% BSA to block) and block for 30 minutes.

[0128] (5) Add primary antibody: Remove the blocking solution, add the prepared primary antibody dropwise, and incubate flat in a humidified chamber at 4°C overnight.

[0129] (6) Add the corresponding secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. Mix the two secondary antibodies and add them dropwise to the circle. Incubate at room temperature in the dark for 50 minutes. Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time.

[0130] (7) Sealing: After adding DAB staining, seal the slides with neutral gum.

[0131] (8) Collect images.

[0132] 2.7 Statistical analysis

[0133] The experimental data were expressed as mean ± standard deviation (mean ± SD) and statistically analyzed using GraphPad Prism 8. One-way ANOVA and two-way ANOVA were used to compare the groups, and P < 0.05 was considered a significant difference.

[0134] 3. Experimental Results

[0135] 3.1 Qipeng Pills (micropills) have a significant anti-inflammatory effect on LPS-induced mouse lungs

[0136] like Figure 3 The results showed that compared with the control group, the levels of inflammatory factors TNF-α and IL-6 in the LPS group were significantly increased. The high-dose group of Qipeng Pills (micropills) significantly inhibited their expression, and the effect of the high-dose group (480mg / kg) was comparable to that of the positive drug.

[0137] 3.2 Qipeng Pills (Micropellets) can improve LPS-induced lung pathological characteristics in mice

[0138] like Figure 4 HE results showed that the alveolar structure of mice in the LPS group was severely damaged or even disappeared, the alveolar wall was thickened, and a large number of inflammatory cells infiltrated the lung tissue. However, the Qipeng Pill (micropill) group was able to significantly reverse this lung inflammation pathology. IHC results showed that the expression levels of inflammatory factors IL-1β, TNF-α, and IL-6 in mice treated with Qipeng Pill (micropill) were significantly reduced.

[0139] These experimental results demonstrate that Qipeng Pills significantly inhibit the expression of inflammatory factors in acute lung injury and reverse lung inflammation. Therefore, Qipeng Pills have the potential to treat acute lung injury and hold great promise for its treatment.

Claims

1. A method for preparing Tibetan medicine Qipeng pills, comprising an active ingredient and microcrystalline fiber, wherein the microcrystalline fiber accounts for 30% of the weight of the pills, and the active ingredient is prepared from fine powders of seven raw materials: Terminalia chebula (coreless), ferox chu (processed), costus root, calamus tatarinowii, myrrh, artificial bezoar, and artificial musk, the method comprising the following steps: 1) uniformly mixing the active component and the microcrystalline fiber to obtain a mixture; 2) Add 75% ethanol as a wetting agent to the mixture to make a soft material; 3) The soft material is fed into an extruder and extruded into a columnar shape, which is then cut into pellets or small pellets of uniform size in a spheronizer; 4) The obtained micropellets or pellets are dried in a fluidized granulation coating machine and sieved.

2. The preparation method according to claim 1, wherein in step 3), the stripping hole diameter is 2 mm.

3. The preparation method according to claim 1, wherein in step 3), the spheronization speed of the spheronizer is 600 rpm.

4. The preparation method according to claim 1, wherein in step 3), the spheronization time of the spheronizer is 3 minutes.

5. The preparation method according to claim 1, wherein in step 4), the drying temperature is not higher than 80°C, and the pellets are dried until the moisture content is ≤8.5%.

6. The preparation method according to claim 1, wherein the active component is prepared from the fine powder of seven raw materials: 400-450 parts of Terminalia chebula (de-cored), 50-60 parts of iron hammer (processed), 60-70 parts of costus root, 350-400 parts of Tibetan calamus, 25-35 parts of myrrh, 5-10 parts of artificial bezoar, and 5-10 parts of artificial musk.

7. The preparation method according to claim 6, wherein the active ingredient is composed of 439.7g of Terminalia chebula (de-cored), 56.4g of iron hammer (processed), 67.4g of costus root, 391.9g of Tibetan calamus, 29.6g of myrrh, 8g of artificial bezoar, 7g of artificial musk, or their weight parts.

8. The preparation method according to any one of claims 1 to 7, wherein the β-asarone content in the Qipeng Pills is not less than 0.8 mg / g.

9. The use of Qipeng Pills in the manufacture of drugs for treating acute lung injury.

10. The use according to claim 8, wherein the Qipeng Pills are prepared by the preparation method according to any one of claims 1 to 7.

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