A traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis and its preparation method and application

Abstract

The present invention discloses a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis, its preparation method, and application. The traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis is prepared by mixing a verbena extract with baicalin nanocrystals and is prepared by conventional methods into various medically acceptable dosage forms, including oral preparations such as tablets, capsules, granules, or oral liquids. The traditional Chinese medicine composition of the present invention can significantly improve bleomycin sulfate-induced idiopathic pulmonary fibrosis in rats, and at the same time alleviate the symptoms of pulmonary fibrosis in rats by reducing the content of inflammatory factors, total protein content, and collagen deposition. It can also improve the oral bioavailability of monomeric baicalin and the traditional Chinese medicine verbena, and is suitable for the prevention and treatment of idiopathic pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis, and a preparation method and use thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung disease with an unknown etiology that predominantly affects middle-aged and elderly individuals. It is characterized by widespread lung remodeling caused by abnormal extracellular matrix deposition, ultimately leading to pulmonary fibrosis. The progression of IPF involves lung damage, inflammation, myofibroblast formation, and accumulation of extracellular matrix, ultimately leading to structural and functional impairment of the lungs. Clinical symptoms are insidious, with early symptoms often presenting as cough and sputum production, followed by worsening dyspnea and eventually respiratory failure and death. Clinically, IPF is difficult to treat, and currently available medications are expensive, suitable for short-term or acute use, and have significant side effects, making them unsuitable for long-term administration. Compared to Western medicine, traditional Chinese medicine (TCM) exhibits less loss of active ingredients, fewer side effects, and the dosage can be adjusted based on individual symptoms. Furthermore, oral TCM preparations are more suitable for treating IPF. Summary of the Invention

[0003] Given the numerous limitations of currently available single-drug therapies, the present invention aims to provide a traditional Chinese medicine composition for the treatment of idiopathic pulmonary fibrosis, thereby improving the situation of patients with the disease. This composition, primarily made from traditional Chinese medicine extracts, features a simple preparation process, significant efficacy, and high safety, making it suitable for the treatment of idiopathic pulmonary fibrosis.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The present invention provides a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis, comprising verbena extract and baicalin nanocrystals (the structural formulas of the main components baicalin, verbenaside and verbenaside are respectively Figure 1 A, B, C in the diagram).

[0006] The total amount of verbenaside and verbenaside in the verbena extract is 3.03% or more, preferably 3.07% or more, and both can be prepared into extracts by water extraction; for example, according to the following method:

[0007] Weigh the verbena herb, crush it, and pass it through a 40-mesh sieve. Add 10-12 times the amount of distilled water and perform reflux extraction twice, each time for 2 hours. After the extraction is completed, combine the extracts, concentrate under reduced pressure, and freeze-dry to obtain the verbena extract. The baicalin nanocrystals are nanoparticles formed by baicalin and poloxamer 188, prepared by a high-pressure homogenization method. The resulting baicalin nanocrystals have a particle size of 330-400 nm. The specific preparation method is as follows: weigh the baicalin raw material, place it in an aqueous solution containing 0.1%-0.2% poloxamer 188, ultrasonically pre-disperse it, and then place it in a high-pressure homogenizer. After 5 cycles at 300 bar, 25 cycles at 1000 bar, obtain a nanocrystal suspension, and freeze-dry it to obtain the baicalin nanocrystals. Preferably, the content of baicalin in the obtained baicalin nanocrystals is 65-85%, preferably 85%; preferably, the weight ratio of verbena extract to baicalin nanocrystals in the composition is 1-5:0.5-2, preferably 2:1, 3:1 or 5:2.

[0008] Furthermore, the present invention also proposes the use of the traditional Chinese medicine composition in preparing a medicine for treating idiopathic pulmonary fibrosis.

[0009] Among them, preferably, the traditional Chinese medicine composition can be prepared into various oral preparations according to conventional traditional Chinese medicine preparation methods, wherein, preferably, the oral preparations include tablets, granules, capsules, and oral liquids.

[0010] Experiments have shown that the traditional Chinese medicine composition of the present invention can significantly improve idiopathic pulmonary fibrosis in rats caused by bleomycin sulfate, and at the same time alleviate the symptoms of pulmonary fibrosis in rats by reducing the content of inflammatory factors, total protein content and collagen deposition, and has potential development value for the prevention and treatment of idiopathic pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The structural formulas of baicalin, verbenaside and verbenaside, where A is baicalin; B is verbenaside; C is verbenaside.

[0012] Figure 2 HE and MASSON staining results (200X), where AH are the HE staining results of rat lung tissue: A blank group; B model group; C positive drug group; D baicalin raw material group; E baicalin nanocrystal group; F verbena extract group; G composition group 1; H composition group 2; IP are the MASSON staining results of rat lung tissue: I blank group; J model group; K positive drug group; L baicalin raw material group; M baicalin nanocrystal group; N verbena extract group; O composition group 1; P composition group 2.

[0013] Figure 3Body weight changes of rats over 28 days.

[0014] Figure 4 MICRO-CT images of rats.

[0015] Figure 5 Rat lung coefficient levels.

[0016] Figure 6 Total protein levels in rat lung homogenates.

[0017] Figure 7 Levels of IL-6, IL-1β, IL-4, and TGF-β1 in rat lung tissue homogenates.

[0018] Figure 8 Lung function level of rats. DETAILED DESCRIPTION

[0019] Example 1

[0020] 1) Weigh verbena, crush it, and pass it through a 40-mesh sieve. Add 10 times the total weight of water and perform reflux extraction. Extract twice, each time for 2 hours. After the extraction is complete, combine the two extracts, concentrate under reduced pressure, and freeze-dry to obtain the verbena extract.

[0021] 2) Baicalin was weighed and placed in an aqueous solution containing 0.1% poloxamer 188. After ultrasonic pre-dispersion, the mixture was placed in a high-pressure homogenizer and subjected to a full cycle of 300 bar for 5 times, followed by 1000 bar for 25 times to obtain a nanocrystal suspension. This suspension was freeze-dried to obtain baicalin nanocrystals with a baicalin content of 65%.

[0022] 3) mixing the verbena extract and baicalin nanocrystals in a weight ratio of 2:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0023] Example 2

[0024] 1) Weigh verbena, crush it, and pass it through a 40-mesh sieve. Add 10 times the total weight of water and perform reflux extraction. Extract twice, each time for 2 hours. After the extraction is complete, combine the two extracts, concentrate under reduced pressure, and freeze-dry to obtain the verbena extract.

[0025] Baicalin was weighed and placed in an aqueous solution containing 0.15% poloxamer 188. After ultrasonic pre-dispersion, the mixture was placed in a high-pressure homogenizer and subjected to a full cycle of 300 bar for 5 times, followed by 1000 bar for 25 times to obtain a nanocrystal suspension. This suspension was freeze-dried to obtain baicalin nanocrystals with a baicalin content of 85%.

[0026] 3) mixing the verbena extract and baicalin nanocrystals in a weight ratio of 2:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0027] Example 3

[0028] 1) Weigh verbena, crush it, and pass it through a 40-mesh sieve. Add 10 times the total weight of water and perform reflux extraction. Extract twice, each time for 2 hours. After the extraction is complete, combine the two extracts, concentrate under reduced pressure, and freeze-dry to obtain the verbena extract.

[0029] 2) Baicalin was weighed and placed in an aqueous solution containing 0.2% poloxamer 188. After ultrasonic pre-dispersion, the mixture was placed in a high-pressure homogenizer and subjected to a full cycle of 300 bar for 5 times, followed by 1000 bar for 25 times to obtain a nanocrystal suspension. This suspension was freeze-dried to obtain baicalin nanocrystals with a baicalin content of 75%.

[0030] 3) mixing the verbena extract and baicalin nanocrystals in a weight ratio of 2:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0031] Example 4

[0032] 1) Weigh verbena, crush it, and pass it through a 40-mesh sieve. Add 10 times the total weight of water and perform reflux extraction. Extract twice, each time for 2 hours. After the extraction is complete, combine the two extracts, concentrate under reduced pressure, and freeze-dry to obtain the verbena extract.

[0033] Baicalin was weighed and placed in an aqueous solution containing 0.15% poloxamer 188. After ultrasonic pre-dispersion, the mixture was placed in a high-pressure homogenizer and subjected to a full cycle of 300 bar for 5 times, followed by 1000 bar for 25 times to obtain a nanocrystal suspension. This suspension was freeze-dried to obtain baicalin nanocrystals with a baicalin content of 85%.

[0034] 3) mixing the verbena extract and baicalin nanocrystals in a weight ratio of 3:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0035] Example 5

[0036] 1) Weigh verbena, crush it, and pass it through a 40-mesh sieve. Add 10 times the total weight of water and perform reflux extraction. Extract twice, each time for 2 hours. After the extraction is complete, combine the two extracts, concentrate under reduced pressure, and freeze-dry to obtain the verbena extract.

[0037] Baicalin was weighed and placed in an aqueous solution containing 0.15% poloxamer 188. After ultrasonic pre-dispersion, the mixture was placed in a high-pressure homogenizer and subjected to a full cycle of 300 bar for 5 times, followed by 1000 bar for 25 times to obtain a nanocrystal suspension. This suspension was freeze-dried to obtain baicalin nanocrystals with a baicalin content of 85%.

[0038] 3) The verbena extract and baicalin nanocrystals are mixed in a weight ratio of 5:2 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0039] Experimental Example 1 Preparation of Verbena Extract

[0040] 1.1 Instruments and reagents

[0041] Constant temperature electric heating mantle (ZDHW, Beijing Zhongxing Weiye Instrument Co., Ltd.); water bath (HH-8, Changzhou Yuexin Instrument Manufacturing Co., Ltd.); rotary evaporator (N-1100, Shanghai Ailang Instrument Co., Ltd.); ultrasonic cleaning machine (KQ-400KDE, Kunshan Ultrasonic Instrument Co., Ltd.); high performance liquid chromatograph (Agilent-1260, Agilent Technologies Co., Ltd.); one-millionth balance (XPR6UD50, Mettler-Toledo); one-tenth balance (AX224ZH, Ohaus Instrument (Changzhou) Co., Ltd.).

[0042] Verbena officinalis (20230903, Beijing Bencao Fangyuan Pharmaceutical Group Co., Ltd.); verbenaside reference substance (AF22052203, ≥98%, Chengdu Aifa Biotechnology Co., Ltd.); verbenaside reference substance (AF21052409, ≥98%, Chengdu Aifa Biotechnology Co., Ltd.); acetonitrile (chromatographic grade, Tianjin Concord Reagent Co., Ltd.); ethanol (analytical grade, Tianjin Concord Reagent Co., Ltd.); phosphoric acid (218128, Thermo Fisher Scientific (China) Co., Ltd.).

[0043] 1.2 Preparation method

[0044] Weigh the verbena herb, crush it, and pass it through a 40-mesh sieve. Add 10 times the total weight of water to perform reflux extraction. Extract twice, each time for 2 hours. After the extraction is completed, combine the two extracts, concentrate under reduced pressure, and then freeze-dry to obtain the verbena extract.

[0045] 1.3 Content determination

[0046] Chromatographic conditions and system suitability tests were conducted using octadecylsilane bonded silica as the packing agent; a 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase; a column temperature of 20°C; and a detection wavelength of 237 nm (for verbenaside and verbenaside). The elution gradient was: 15% B (0-3 min); 15%-25% B (3-8 min); 25%-30% B (8-11 min); and 30% B (11-15 min).

[0047] Preparation of reference solution: 8.92 mg and 10.82 mg of verbenaside and verbenaside reference substances were accurately measured, placed in a 5 mL volumetric flask and dissolved in 50% methanol to obtain mixed reference solution with concentrations of 1.784 mg / mL and 2.6144 mg / mL, respectively.

[0048] Preparation of the test solution: Take 20 mg of the extract obtained by freeze-drying, accurately add 20 ml of 50% methanol, ultrasonicate for 40 minutes, filter, and take the filtrate to obtain the solution.

[0049] The total amount of verbenaside and verbenaside in the prepared verbena extract was determined to be 3.03%-3.07%.

[0050] Experimental Example 2 Preparation of Baicalin Nanocrystals

[0051] 2.1 Instruments and reagents

[0052] High-pressure homogenizer (AH00D, ATS Industrial Systems Co., Ltd.); Malvern particle size and potential analyzer (Nano-ZS, Marlvern); ultrasonic cleaning machine (KQ-400KDE, Kunshan Ultrasonic Instrument Co., Ltd.); high-performance liquid chromatograph (Agilent-1260, Agilent Technologies Co., Ltd.); one-millionth balance (XPR6UD50, Mettler-Toledo); one-tenth balance (AX224ZH, Ohaus Instruments (Changzhou) Co., Ltd.).

[0053] Baicalin (JS252432, ≥90%, Shanghai Yuanye Biotechnology Co., Ltd.); baicalin reference substance (N15GB167969, ≥98%, Shanghai Yuanye Biotechnology Co., Ltd.); poloxamer (BASF Co., Ltd.); acetonitrile (chromatographic grade, Tianjin Concord Reagent Co., Ltd.); ethanol (analytical grade, Tianjin Concord Reagent Co., Ltd.); phosphoric acid (218128, Thermo Fisher Scientific (China) Co., Ltd.).

[0054] 2.2 Preparation method

[0055] The baicalin raw material was weighed and placed in an aqueous solution containing 0.15% poloxamer 188. After ultrasonic pre-dispersion, it was placed in a high-pressure homogenizer. After 5 full cycles at 300 bar and 25 cycles at 1000 bar, a nanocrystal suspension was obtained with a measured particle size of 350 nm. Baicalin nanocrystals were obtained by freeze-drying. After re-dissolution, the particle size was measured to be 370 nm, with no significant change.

[0056] 2.3 Content determination

[0057] Chromatographic conditions and system suitability were tested using octadecylsilane bonded silica gel as the packing material; a 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) mobile phase; a column temperature of 20°C; and a detection wavelength of 237 nm (for baicalin). The elution gradient was: 15% B (0-3 min); 15%-25% B (3-8 min); 25%-30% B (8-11 min); and 30% B (11-15 min).

[0058] Preparation of reference solution: Accurately measure 5.50 mg of baicalin reference substance, place it in a 5 mL volumetric flask and dissolve it in methanol to obtain a mixed reference solution with a concentration of 1.10 mg / mL.

[0059] Preparation of the test solution: Take 5 mg of the nanocrystals obtained by freeze-drying, accurately add 25 ml of methanol, ultrasonicate for 10 minutes, and take the filtrate to obtain the solution.

[0060] It was determined that the baicalin content in the prepared baicalin nanocrystals was 84.54%.

[0061] Experimental Example 3 Preparation of Chinese medicine composition

[0062] According to the weight ratio of verbena extract to baicalin nanocrystals being 2:1, the verbena extract and baicalin nanocrystals prepared in Experimental Example 1 and Experimental Example 2 were weighed and mixed to obtain a verbena extract-baicalin nanocrystal composition.

[0063] Experimental Example 4 Pharmacodynamic Study of the Traditional Chinese Medicine Composition Prepared in Experimental Example 3 on Idiopathic Pulmonary Fibrosis in Rats

[0064] 4.1 Experimental Animals and Test Drugs

[0065] 4.1.1 Trial Drugs

[0066] Verbena extract (prepared according to Experimental Example 1); baicalin nanocrystals (prepared according to Experimental Example 2); verbena extract-baicalin nanocrystal composition 1 (prepared according to Experimental Example 3, 90 mg / kg); verbena extract-baicalin nanocrystal composition 2 (prepared according to Experimental Example 3, 180 mg / kg); physiological saline (G4702, Wuhan Servicebio Company); 4% tissue fixative (P1110, Beijing Solebow Technology Co., Ltd.).

[0067] Pirfenidone capsules (National Medicine Standard No. H20133376, Beijing Contini Pharmaceutical Co., Ltd.); bleomycin sulfate (B107423-100 mg, Shanghai Aladdin Biochemical Technology Co., Ltd.); rat BCA protein quantitative detection kit (060723240430, Shanghai Biyuntian Biotechnology Co., Ltd.); rat interleukin-6 (IL-6), rat interleukin-1β (IL-1β), rat interleukin-4 (IL-4), and rat transforming growth factor (TGF-β1) ELISA kits (MM-0190R1, MM-0047R1, MM-0191R1, MM-0181R1, Jiangsu Enzyme Immunity Industry Co., Ltd.); trichloraldehyde hydrate (302-17-0, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0068] 4.1.2 Experimental animals

[0069] 80 male SD rats weighing 180-220 g

[0070] 4.2 Experimental Methods

[0071] 4.2.1 Establishment of rat pulmonary fibrosis model

[0072] A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin sulfate. After anesthesia, rats were placed supine on a 35° tilted board. The neck skin was routinely disinfected with 75% medical alcohol. A 2- to 3-cm midline skin incision was then made, and the muscle was bluntly dissected with forceps to expose the trachea. Next, a No. 4 needle was inserted through the interstices between the tracheal cartilage rings toward the heart, and approximately 0.3 mL of a solution containing bleomycin sulfate (5 mg / kg) was injected through an empty syringe. After the injection, the board was quickly rotated vertically and left upright for 3 minutes to allow for even distribution of the drug in the lungs. The muscle and skin were then sutured and disinfected again. Rats in the blank control group received only an equal volume of normal saline intratracheally; all other procedures were the same as those in the modeling group. All rats except the blank control group were established with a pulmonary fibrosis model.

[0073] 4.2.2 Animal grouping and treatment

[0074] After 3 days of adaptive feeding, 80 SD rats were randomly divided into 8 groups: blank group (Blank), model group (Model), pirfenidone capsule positive drug group (PFD, 52 mg / kg), baicalin raw material group (60 mg / kg), baicalin nanocrystal group (60 mg / kg), verbena extract group (VE, 270 mg / kg), verbena extract-baicalin nanocrystal combination 1 (BA-VE-s, 90 mg / kg) and verbena extract-baicalin nanocrystal combination 2 group (BA-VE-m, 180 mg / kg).

[0075] 4.2.3 Sample collection and storage

[0076] The rats' respiration, activity, food intake, and body weight were observed daily. The bleomycin sulfate model was considered the first day, and body weights were recorded on days 1, 7, 14, 21, and 28 for statistical analysis. Micro-CT imaging was performed on day 28 of the model. Various respiratory parameters, including inspiratory duration (TI), expiratory duration (TE), respiratory rate (f), peak inspiratory flow (PIF), peak expiratory flow (PEF), tidal volume (TV), relaxation time (RT), and bronchoconstriction index (Penh), were measured in freely moving rats using an EMKA pulmonary function tester. After the tests, blood was drawn from the abdominal aorta and the rats were sacrificed. Bronchoalveolar lavage fluid was collected, and lung tissue was removed and weighed. The left lung was fixed in 10% neutral formalin and stained with hematoxylin and eosin (HE) and Masson staining 24 hours later to observe lung pathology.

[0077] 4.2.4 Rat lung imaging examination

[0078] In vivo micro CT scans were performed using a Quantum FX scanner with the following parameters: tube voltage: 90 kV, tube current: 88 μA, imaging field of view: 72 x 40 mm, and reconstructed image pixel size: 144 μm. Whole-lung micro CT images of the rat lungs were obtained with rats in the prone position with their limbs spread out and head forward, anesthetized with isoflurane, and allowed to breathe freely.

[0079] 4.2.5 Rat lung coefficient determination

[0080] Take the lung tissue sample from "4.2.3" and calculate the lung coefficient using the following formula and perform statistical analysis:

[0081] Lung coefficient = lung wet weight (g) / body weight (kg) * 100%

[0082] 4.2.6 Determination of total protein in rat lung tissue homogenate

[0083] Take the lung tissue homogenate sample from "4.2.3" and centrifuge it at 4°C and 4000 rpm for 10 min to obtain the supernatant. Determine the total protein content using a colorimetric method according to the instructions in the total protein assay kit.

[0084] 4.2.7 Determination of IL-6, IL-1β, IL-4, and TGF-β1 Contents in Rat Lung Tissue Homogenates

[0085] Take the lung tissue homogenate sample from "4.2.3" and centrifuge it at 4000 rpm at 4°C for 10 minutes. Obtain the supernatant. Analyze the levels of inflammatory factors IL-4, IL-6, TNF-β1, and IL-1β using enzyme-linked immunosorbent assay (ELISA). Specific experimental procedures should be performed according to the instructions in the relevant ELISA kit.

[0086] 4.2.8 Statistics and Plotting

[0087] The experimental data were analyzed by one-way ANOVA using SPSS 27 software, and the graphics were plotted using Graphpad Prism 8.0.

[0088] 4.3 Experimental Results

[0089] 4.3.1 Observation of rat physiological status

[0090] The rats in the blank group were in good mental state, with stable breathing, regular diet, shiny fur, and no coughing or nasal secretions. The rats in the model group were breathing rapidly, in poor mental state, had severe hair loss, coughing and nasal secretions, and their food intake decreased. The different drug-administered groups showed varying degrees of improvement compared to the model group. As the drug-administered time increased, the rats' breathing gradually stabilized, nasal secretions decreased, and their diet, hair loss, and mental state recovered to varying degrees. Figure 3 As shown, on day 7, the model group showed a significant decrease in body weight compared to the blank group (***p<0.001). On day 14, the pirfenidone, Composition 1, and Composition 2 groups began to show a significant increase in body weight compared to the model group (#p<0.05, ##p<0.01), and this trend was maintained until day 28. On day 21, the baicalin nanocrystal group showed a significant increase in body weight compared to the baicalin API group (▽p<0.05), and this trend was maintained until day 28. On day 28, the body weight of the rats in the Composition 2 group was significantly increased compared to the baicalin nanocrystal group and the verbena extract group (▲p<0.05). The results indicate that baicalin nanocrystals can improve pulmonary fibrosis in rats more than the API, and the combination with verbena is more advantageous than either extract administered alone, significantly improving the weight loss caused by pulmonary fibrosis in rats.

[0091] 4.3.2 Staining of rat lung tissue sections

[0092] HE-stained and MASSON-stained sections of rat lung tissue were obtained and observed under an optical microscope.

[0093] HE staining results showed that the blank group ( Figure 2 A) The lung tissue capsule structure is clear, and the lung substance is composed of the branches of the bronchial tubes at all levels and a large number of alveoli at their terminals, without collagen fiber proliferation; Model group ( Figure 2 B) in the figure shows patchy alveolar consolidation, unclear alveolar structure, numerous lymphocytes, granulocytes and macrophages (green arrows) in the alveolar wall and alveolar cavity, and occasional necrotic cell fragments (black arrows); occasional alveolar dilatation (dark blue arrows); occasional focal lymphocyte infiltration around blood vessels and bronchioles (blue arrows); occasional perivascular edema (purple arrows), loosely arranged connective tissue, widened spaces, with a small amount of punctate lymphocyte infiltration; occasional hydropic degeneration of bronchiolar epithelial cells (red arrows), swollen cells, and loose and lightly stained cytoplasm; positive drug group ( Figure 2 C) The lung tissue capsule structure is clear, and the lung substance is composed of a large number of alveoli at the ends of the bronchial branches at all levels. The alveolar wall is composed of a single layer of epithelium. Focal alveolar wall capillary congestion (orange arrows), rare vascular congestion (orange arrows), and a small amount of brown-yellow pigment deposition on the alveolar wall (brown arrows) show mild lesions; Baicalin API group ( Figure 2 D) A small amount of granulocyte and mast cell infiltration in the alveolar wall (green arrow); multifocal moderate thickening of the alveolar wall and widening of the alveolar septum; a small amount of foam cells in the alveoli and bronchioles (dark red arrow); many alveolar dilatation (dark blue arrow); occasional hydropic degeneration of bronchiolar epithelial cells (red arrow), cell swelling, loose and lightly stained cytoplasm; occasional brown-yellow pigment deposition (brown arrow), showing mild lesions; Baicalin nanocrystal group ( Figure 2 E) A small number of foam cells and mast cells (dark red arrows) in the alveoli, occasional perivascular edema (purple arrows), loose connective tissue arrangement, widened spaces, with a small amount of lymphocyte punctate infiltration, a small amount of eosinophilic tissue fluid (gray arrows), more vascular congestion (orange arrows), occasional brown-yellow pigment deposition (brown arrows), showing mild lesions; Verbena extract group ( Figure 2 F) Infiltration of alveolar walls and alveolar cavities by numerous granulocytes, macrophages, and mast cells (green arrows), moderate thickening of alveolar walls in a small area, widening of alveolar septa, a small amount of necrotic cell debris in the alveoli (black arrows), rare vascular congestion (orange arrows), a small amount of brown-yellow pigment deposition (brown arrows), and occasional hyperplasia of bronchiolar epithelium (light green arrows) were observed, showing mild lesions; Composition 1 ( Figure 2In group G, there was a small amount of granulocyte infiltration in the alveolar wall (green arrows), mild thickening of the alveolar wall in multiple foci, widening of the alveolar septa, a small amount of macrophages in the alveoli (dark red arrows), and a large number of alveolar expansion (dark blue arrows). The bronchiolar epithelial cells were arranged irregularly (light green arrows), and a small amount of epithelial cells were visible in the cavity, showing mild lesions. The combination group 2 ( Figure 2 H) The lung tissue capsule structure is clear. The lung parenchyma consists of numerous alveoli at the ends of the bronchial branches at all levels. The alveolar walls are composed of a single layer of epithelium. There are many foam cells (dark red arrows) in the alveoli, and few vascular congestion (orange arrows), indicating a mild lesion.

[0094] The results of MASSON staining showed that the blank group ( Figure 2 No obvious collagen fiber proliferation was observed in the lung tissue of the model group ( Figure 2 J) Multifocal proliferation of collagen fibers (yellow arrows) was observed in lung tissue, with small fibrous masses formed in many places. The fibers were uneven in thickness and arranged in disorder. Figure 2 In the K) of the lung tissue, a small amount of collagen fiber proliferation (yellow arrow) can be seen locally. The collagen fibers are thin, discontinuous and irregularly arranged. Figure 2 L) Multifocal proliferation of collagen fibers (yellow arrows) was observed in lung tissue, with small fiber masses formed in many places. The fibers were uneven in thickness and arranged in disorder. Baicalin nanocrystal group ( Figure 2 M in the figure) Multifocal proliferation of collagen fibers (yellow arrows) was observed in lung tissue. The collagen fibers were thin, discontinuous and irregularly arranged. Verbena extract group ( Figure 2 Multiple collagen fiber proliferations (yellow arrows) can be seen in the lung tissue of composition 1 ( Figure 2 A small amount of collagen fiber proliferation (yellow arrow) can be seen in the O) of the lung, and the lung tissue is intact; Composition 2 ( Figure 2 A small amount of collagen fiber proliferation is occasionally seen in P), and the collagen fibers are thin.

[0095] The severity of lesions: model group > verbena extract group > baicalin raw material group > composition group 1 > baicalin nanocrystal group > positive drug group > composition group 2.

[0096] 4.3.3 Micro-CT images of rat lungs

[0097] The blank group showed uniform lung parenchyma density on both sides, with clear and evenly distributed lung textures. From the inside to the outside of the lungs, the bronchial and vascular bundles gradually became thinner, with regular patterns forming texture shadows. No abnormal high-density shadows were found in the bilateral lung fields. The model group showed increased lung parenchyma density, blurred lung textures, large nodular or mass-like shadows, disordered lung structure, and abnormal lung interstitial structure. The other drug-treated groups showed varying degrees of improvement compared with the model group. Figure 4 It can be seen that the severity of fibrosis is as follows: model group > verbena extract group > baicalin raw material group > baicalin nanocrystal group > composition group 1 > positive drug group > composition group 2.

[0098] 4.3.4 Rat lung coefficient levels

[0099] The lung coefficients of rats in different groups were measured and the expression of pulmonary edema in the lung tissues of rats in different groups was observed. Figure 5 As shown, the lung coefficient of the model group was significantly increased compared with the blank group (n=10, ***p<0.001), and was significantly decreased in the positive drug group, baicalin raw material group, baicalin nanocrystal group, composition 1 group and composition 2 group compared with the model group (n=10, #p<0.05, ##p<0.01); compared with the baicalin raw material group, the lung coefficient of the baicalin nanocrystal group was significantly decreased (n=10, ▽p<0.05); compared with the baicalin nanocrystal group and verbena extract groups, the lung coefficient of the composition 2 group was significantly decreased (n=10, ▲p<0.05, △p<0.05).

[0100] 4.3.5 Total protein levels in rat lung tissue homogenates

[0101] The total protein content in the bronchoalveolar lavage fluid of rats in each group was determined by BCA method. Figure 6 As shown, the total protein content in the blank group was (7.99±1.50) mg / mL, and the total protein content in the model group was (17.11±1.34) mg / mL. The model group was significantly increased compared with the blank group (n=10, ***p<0.001). Compared with the model group, the total protein content in the positive drug group, baicalin nanocrystal group, verbena extract group, composition 1, and composition 2 groups was significantly decreased (n=10, #p<0.05, ##p<0.01). Compared with the verbena extract group, the total protein content in the composition 2 group was significantly decreased (n=10, △p<0.05).

[0102] 4.3.6 Determination of IL-4, IL-6, IL-1β and TGF-β1 Contents in Rat Lung Tissue Homogenates

[0103] The release and expression of IL-4, IL-6, IL-1β and TGF-β1 in lung tissue homogenates of rats in different groups were measured. Figure 7As shown, the contents of IL-4, IL-6, IL-1β and TGF-β1 in the model group were significantly increased compared with those in the blank group (n=10, ***p<0.001); the contents of IL-4, IL-6, IL-1β and TGF-β1 in the positive drug group, baicalin raw material group, baicalin nanocrystal group, composition 1 group and composition 2 group were significantly decreased compared with those in the model group (n=10, #p<0.05, ##p<0.01, ###p<0.001); compared with the baicalin raw material group, the contents of IL-4, IL-6 and TGF-β1 in the baicalin nanocrystal group were significantly decreased (n=10, ▽p<0.05, ▽▽p<0.01) Compared with the baicalin nanocrystals and verbena extract groups, the levels of IL-4, IL-6 and TGF-β1 in composition 2 were significantly reduced (n=10, ▲p<0.05, △p<0.05).

[0104] 4.3.7 Rat lung function level

[0105] from Figure 8 It can be seen from the pulmonary function indicators that compared with the blank group, the pulmonary function parameters of the model group rats Ti (inspiratory duration), Te (expiratory duration), PIF (maximum inspiratory flow), TV (tidal volume), MV (minute volume), and RT (relaxation time) were significantly reduced, and Penh (bronchial contraction parameter) and f (respiratory frequency) were significantly increased (n=10, ***p<0.001); the respiratory indicators of the drug-treated group showed significant improvement compared with the model group (n=10, #p<0.05, ##p<0.01, ###p<0.001); except for Te, the other pulmonary function indicators of the baicalin nanocrystal group showed significant improvement compared with the baicalin raw material group (n=10, ▽p<0.05, ▽▽p<0.01); Compared with the baicalin nanocrystal group and the verbena extract group, the combination group 2 showed significant increases in Ti, Te, PEF, MV, Penh and f (n=10, △p<0.05, ▲p<0.05, ▲▲p<0.01).

[0106] The above experimental results show that both baicalin and verbena extract alone can alleviate the symptoms of pulmonary fibrosis in rats by reducing the levels of inflammatory factors, total protein content, and collagen deposition, and have a certain therapeutic effect on the treatment of pulmonary fibrosis. When baicalin is made into nanocrystals, its efficacy is also better than that of the raw material in terms of various indicators of idiopathic pulmonary fibrosis. The combination of baicalin nanocrystals and verbena extract also showed significant differences compared with the two single drugs, indicating that the combination of the two drugs is more effective.

Claims

1. Use of a traditional Chinese medicine composition consisting of verbena extract and baicalin nanocrystals in preparing a medicament for treating idiopathic pulmonary fibrosis, characterized in that: The verbena extract is prepared according to the following method: taking verbena herbs, crushing them, and then passing them through a 40-mesh sieve, adding 10-12 times the amount of distilled water for reflux extraction, and extracting them twice, each time for 2 hours; after the extraction is completed, combining the extracts, concentrating under reduced pressure, and then freeze-drying to obtain the verbena extract; the mass ratio of verbena extract to baicalin nanocrystals in the traditional Chinese medicine composition is 1-5:0.5-2.

2. The use according to claim 1, characterized in that The total amount of verbenaside and verbenaside in the verbena extract is more than 3.03%.

3. The use according to claim 1 or 2, characterized in that The content of baicalin in baicalin nanocrystals is 65%-85%.

4. The use according to claim 1 or 2, characterized in that The baicalin nanocrystals are nanoparticles prepared by high-pressure homogenization of baicalin in a poloxamer 188 aqueous solution with a mass percentage concentration of 0.1%-0.2%.

5. The use according to claim 4, characterized in that The mass percentage concentration of the poloxamer 188 aqueous solution is 0.1%.

6. The use according to claim 4, characterized in that The mass percentage concentration of the poloxamer 188 aqueous solution is 0.15%.

7. The use according to claim 4, characterized in that The mass percentage concentration of the poloxamer 188 aqueous solution is 0.2%.

8. The use according to claim 1 or 2, characterized in that The mass ratio of the verbena extract to the baicalin nanocrystals is 2:

1.

9. The use according to claim 1 or 2, characterized in that The mass ratio of the verbena extract to the baicalin nanocrystals is 3:

1.

10. The use according to claim 1 or 2, characterized in that The mass ratio of the verbena extract to the baicalin nanocrystals is 5:

2.

11. The use according to claim 4, characterized in that The preparation method of baicalin nanocrystals comprises the following steps: (1) Weigh baicalin, place it in a poloxamer 188 aqueous solution, and pre-disperse it by ultrasonication; (2) The pre-dispersed mixture was added to a high-pressure homogenizer and circulated 5 times at 300 bar and 25 times at 1000 bar to obtain a nanocrystal suspension; (3) Freeze-drying the nanocrystal suspension to obtain baicalin nanocrystals.

12. The use according to claim 1 or 2, characterized in that The particle size of the baicalin nanocrystals is 330-400nm.

13. The use according to claim 1 or 2, characterized in that: The Chinese medicine composition is prepared into an oral preparation.

14. The use according to claim 13, characterized in that: Oral preparations include tablets, granules, capsules, and oral solutions.

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