Traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis and preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis and a preparation method and application thereof. The traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis is formed by mixing a verbena extract and baicalin nanocrystals, and various medically acceptable dosage forms including tablets, capsules, granules or oral liquid and other oral preparations are prepared through a conventional method. The traditional Chinese medicine composition disclosed by the invention can be used for remarkably improving rat idiopathic pulmonary fibrosis caused by bleomycin sulfate, relieving the symptom of rat pulmonary fibrosis by reducing the inflammatory factor content, the total protein content and collagen deposition, and improving the oral bioavailability of the monomer baicalin and the traditional Chinese medicine verbena; the traditional Chinese medicine composition is suitable for preventing and treating idiopathic pulmonary fibrosis.

Description

Technical Field

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

[0002] Idiopathic pulmonary fibrosis is a chronic and irreversible diffuse alveolitis and alveolar structural disorder with unclear etiology, which is prone to occur in middle-aged and elderly people. It is characterized by extensive lung remodeling caused by abnormal deposition of extracellular matrix, and eventually leads to pulmonary interstitial fibrosis. The development of idiopathic pulmonary fibrosis includes lung damage, inflammation, the formation of myofibroblasts and the accumulation of extracellular matrix, which eventually leads to pulmonary structural dysfunction. The clinical symptoms are insidious onset, and the early symptoms are not obvious. Initially, they present as cough and sputum, and later, dyspnea worsens, leading to respiratory failure and death. In clinical practice, it is found that idiopathic pulmonary fibrosis is difficult to treat, and the drugs used are expensive. They are all suitable for short-term or acute medication, with large toxic and side effects, and are not suitable for long-term administration. Compared with Western medicine, the effective ingredients of traditional Chinese medicine are not easy to dissipate, the side effects are small, and they can be added or subtracted at any time according to different symptoms. And the range of diseases treated is wider. Therefore, oral preparations of traditional Chinese medicine are more suitable for improving idiopathic pulmonary fibrosis. Summary of the invention

[0003] In view of the many limitations of the single drugs currently on the market, the present invention aims to provide a Chinese medicine composition for treating idiopathic pulmonary fibrosis to improve the situation of patients with idiopathic pulmonary fibrosis. The drug of the present invention uses Chinese medicine extracts as the main raw materials, has a simple preparation process, significant efficacy, high safety, and is suitable for the treatment of idiopathic pulmonary fibrosis.

[0004] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a Chinese medicine composition for treating idiopathic pulmonary fibrosis, comprising a verbena extract and baicalin nanocrystals (the structural formulas of the main components baicalin, verbena glycoside and verbena glycoside are respectively Figure 1 A, B, C in the figure).

[0005] 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 extracted into extracts by water; for example, they can be prepared by the following method: Weigh the verbena material, crush it and pass it through a 40-mesh sieve, add 10-12 times the amount of distilled water for reflux extraction, extract 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. Wherein, the baicalin nanocrystals are nanoparticles formed by baicalin and poloxamer 188, prepared by a high-pressure homogenization method, and the obtained baicalin nanocrystals have a particle size of 330-400nm. The specific preparation method is: weigh the baicalin raw material, place it in an aqueous solution containing 0.1%-0.2% poloxamer 188, ultrasonically pre-disperse it, place it in a high-pressure homogenizer, circulate it at 300bar for 5 times, and circulate it at 1000bar for 25 times to obtain a nanocrystal suspension, and freeze-dry it to obtain 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.

[0006] Furthermore, the present invention also proposes the use of the traditional Chinese medicine composition in preparing a medicine for treating idiopathic pulmonary fibrosis.

[0007] Among them, preferably, the Chinese medicine composition can be prepared into various oral preparations according to conventional Chinese medicine preparation methods, wherein, preferably, the oral preparations include tablets, granules, capsules, and oral liquids.

[0008] Experiments have shown that the 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

[0009] Figure 1 The structural formulas of baicalin, verbenaside and verbenaside, wherein A is baicalin; B is verbenaside; C is verbenaside.

[0010] 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.

[0011] Figure 3Body weight changes of rats over 28 days.

[0012] Figure 4 MICRO-CT images of rats.

[0013] Figure 5 Rat lung coefficient levels.

[0014] Figure 6 Total protein levels in rat lung homogenates.

[0015] Figure 7 Levels of IL-6, IL-1β, IL-4, and TGF-β1 in rat lung tissue homogenates.

[0016] Figure 8 Lung function levels in rats. DETAILED DESCRIPTION

[0017] Example 1 1) Weigh verbena, 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 freeze-dry to obtain verbena extract; 2) Baicalin was weighed and placed in an aqueous solution containing 0.1% poloxamer 188. After ultrasonic pre-dispersion, it was placed in a high-pressure homogenizer. After 5 cycles at 300 bar, it was cycled 25 times at 1000 bar to obtain a nanocrystal suspension. The baicalin nanocrystals were freeze-dried to obtain baicalin nanocrystals. The baicalin content in the nanocrystals was 65%; 3) The verbena extract and baicalin nanocrystals are mixed in a weight ratio of 2:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0018] Example 2 1) Weigh verbena, 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 freeze-dry to obtain verbena extract; 2) Baicalin 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 cycles at 300 bar, it was cycled 25 times at 1000 bar to obtain a nanocrystal suspension. The baicalin nanocrystals were freeze-dried to obtain baicalin nanocrystals. The baicalin content in the nanocrystals was 85%; 3) The verbena extract and baicalin nanocrystals are mixed in a weight ratio of 2:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0019] Example 3 1) Weigh verbena, 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 freeze-dry to obtain verbena extract; 2) Baicalin was weighed and placed in an aqueous solution containing 0.2% poloxamer 188. After ultrasonic pre-dispersion, it was placed in a high-pressure homogenizer. After 5 cycles at 300 bar, it was cycled 25 times at 1000 bar to obtain a nanocrystal suspension. The baicalin nanocrystals were freeze-dried to obtain baicalin nanocrystals. The baicalin content in the nanocrystals was 75%; 3) The verbena extract and baicalin nanocrystals are mixed in a weight ratio of 2:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0020] Example 4 1) Weigh verbena, 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 freeze-dry to obtain verbena extract; 2) Baicalin 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 cycles at 300 bar, it was cycled 25 times at 1000 bar to obtain a nanocrystal suspension. The baicalin nanocrystals were freeze-dried to obtain baicalin nanocrystals. The baicalin content in the nanocrystals was 85%; 3) The verbena extract and baicalin nanocrystals are mixed in a weight ratio of 3:1 to obtain a traditional Chinese medicine composition for treating idiopathic pulmonary fibrosis.

[0021] Example 5 1) Weigh verbena, 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 freeze-dry to obtain verbena extract; 2) Baicalin 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 cycles at 300 bar, it was cycled 25 times at 1000 bar to obtain a nanocrystal suspension. The baicalin nanocrystals were freeze-dried to obtain baicalin nanocrystals. The baicalin content in the nanocrystals was 85%; 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.

[0022] Experimental Example 1 Preparation of Verbena Extract 1.1 Instruments and reagents 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-millionth balance (AX224ZH, Ohaus Instrument (Changzhou) Co., Ltd.).

[0023] Verbena (20230903, Beijing Bencao Fangyuan Pharmaceutical Group Co., Ltd.); verbena glycoside reference substance (AF22052203, ≥98%, Chengdu Aifa Biotechnology Co., Ltd.); verbena glycoside 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.).

[0024] 1.2 Preparation method Weigh the verbena medicinal material, crush it and pass it through a 40-mesh sieve, add 10 times the total weight of water for reflux extraction, extract twice, each time for 2 hours. After the extraction is completed, combine the two extracts, concentrate under reduced pressure and freeze-dry to obtain the verbena extract.

[0025] 1.3 Content determination Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; 0.1% phosphoric acid water (A)-acetonitrile (B) solution was used as mobile phase; column temperature was 20°C; detection wavelength was 237nm (verbenin, verbenaside). The elution gradient was 0-3min, 15%B; 3-8min, 15%-25%B; 8-11min, 25%-30%B; 11-15min, 30%B.

[0026] Preparation of reference solution: 8.92 mg and 10.82 mg of verbena glycoside and verbena glycoside reference substances were accurately measured respectively, placed in a 5 mL volumetric flask and dissolved in 50% methanol to obtain a mixed reference solution with concentrations of 1.784 mg / mL and 2.6144 mg / mL respectively.

[0027] 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 min, filter, and take the filtrate to obtain the test solution.

[0028] It was determined that the total amount of verbenaside and verbenaside in the verbena extract was 3.03%-3.07%.

[0029] Experimental Example 2 Preparation of Baicalin Nanocrystals 2.1 Instruments and reagents 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 Instrument (Changzhou) Co., Ltd.).

[0030] 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.).

[0031] 2.2 Preparation method 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 nanocrystalline 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 obvious change.

[0032] 2.3 Content determination Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; 0.1% phosphoric acid water (A)-acetonitrile (B) solution was used as mobile phase; column temperature was 20°C; detection wavelength was 237nm (baicalin). The elution gradient was 0-3min, 15%B; 3-8min, 15%-25%B; 8-11min, 25%-30%B; 11-15min, 30%B.

[0033] 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.

[0034] Preparation of the test solution: Take 5 mg of the nanocrystals obtained by freeze-drying, accurately add 25 ml of methanol, ultrasonicate for 10 min, and take the filtrate to obtain the test solution.

[0035] It was determined that the content of baicalin in the prepared baicalin nanocrystals was 84.54%.

[0036] Experimental Example 3 Preparation of Chinese medicine composition 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.

[0037] Experimental Example 4 Pharmacodynamic study of the traditional Chinese medicine composition prepared in Experimental Example 3 on idiopathic pulmonary fibrosis in rats 4.1 Experimental Animals and Test Drugs 4.1.1 Drug testing 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.).

[0038] Pirfenidone capsules (National Medicine Standard No. H20133376, Beijing Contini Pharmaceutical Co., Ltd.); Bleomycin sulfate (B107423-100mg, 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), rat transforming growth factor (TGF-β1) ELISA kits (MM-0190R1, MM-0047R1, MM-0191R1, MM-0181R1, Jiangsu Enzyme Immunity Industry Co., Ltd.); Chloraldehyde hydrate (302-17-0, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0039] 4.1.2 Experimental animals 80 male SD rats weighing 180-220 g 4.2 Experimental methods 4.2.1 Establishment of rat pulmonary fibrosis model The rat pulmonary fibrosis model was established by intratracheal instillation of bleomycin sulfate. After anesthetizing the rats, they were fixed on a board tilted at 35° in a supine position, and the neck skin was routinely disinfected with 75% medical alcohol. Then, a 2 to 3 cm skin incision was made in the midline of the neck, and the muscles were bluntly separated using vascular clamps to expose the trachea. Next, a No. 4 needle was used to penetrate the gap between the tracheal cartilage rings, puncture the trachea toward the heart, and inject about 0.3 mL of a solution containing bleomycin sulfate (at a dose of 5 mg / kg) through an empty syringe. After the injection, the mouse board was quickly rotated vertically and placed upright for 3 min to allow the drug to be evenly distributed in the lungs. After that, the muscles and skin were sutured and disinfected again. The blank control group rats only received an intratracheal injection of an equal amount of normal saline, and other operations were the same as those of the modeling group. Except for the blank control group, all other groups were made into pulmonary fibrosis models.

[0040] 4.2.2 Animal grouping and treatment 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).

[0041] 4.2.3 Sample collection and storage The rats' breathing, activity, eating and body weight were observed daily. The bleomycin sulfate model was regarded as the first day. The body weights of the rats in each group were recorded on days 1, 7, 14, 21 and 28, and statistical analysis was performed. MICRO-CT imaging examination of rats was performed on day 28 of modeling, and various respiratory indicators of rats in free-moving state were tested with EMKA pulmonary function tester, including duration of inspiration (TI), duration of expiration (TE), respiratory rate (f), maximum inspiratory flow (PIF), maximum expiratory flow (PEF), tidal volume (TV), relaxation time (RT), bronchial contraction parameter (Penh), etc. After the test, blood was collected from the abdominal aorta and the rats were killed. The alveolar lavage fluid of the rats was collected, and the lung tissue was removed and weighed. The left lung was immersed in 10% neutral formalin fixative, and HE staining and MASSON staining were performed 24 hours later to observe the lesions of lung tissue.

[0042] 4.2.4 Rat lung imaging examination The model of in vivo Micro CT scanning was Quantum FX, and the scanning parameters were as follows: tube voltage: 90kV, tube current: 88uA, imaging field of view: 72*40 mm, pixel size of reconstructed image: 144 um. The in vivo rats were placed in prone position with their limbs spread out and head forward, anesthetized with isoflurane, and whole lung scans were performed under free breathing to obtain micro CT images of the rat lungs.

[0043] 4.2.5 Rat lung coefficient determination Take the lung tissue sample in "4.2.3" and calculate the lung coefficient using the following formula and perform statistical analysis: Lung coefficient = lung wet weight (g) / body weight (kg) * 100% 4.2.6 Determination of total protein in rat lung tissue homogenate Take the lung tissue homogenate sample in "4.2.3" and centrifuge it at 4°C and 4000 rpm for 10 min to obtain the supernatant. Determine the total protein content by colorimetry according to the instructions of the total protein determination kit.

[0044] 4.2.7 Determination of IL-6, IL-1β, IL-4 and TGF-β1 contents in rat lung tissue homogenates Take the lung tissue homogenate sample in "4.2.3" and centrifuge it at 4°C and 4000 rpm for 10 min to obtain the supernatant. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory factors IL-4, IL-6, TNF-β1, and IL-1β. The specific experimental operation was carried out according to the steps in the instructions of the relevant ELISA kit.

[0045] 4.2.8 Statistics and Plotting The experimental data were analyzed by one-way ANOVA using SPSS 27 software and plotted using Graphpad Prism 8.0.

[0046] 4.3 Experimental Results 4.3.1 Observation of rat physiological status The rats in the blank group had a good mental state, stable breathing, regular diet, shiny fur, and no coughing or nasal secretions. The rats in the model group had rapid breathing, poor mental state, severe hair loss, coughing and nasal secretions, and reduced food intake. The different drug-administered groups showed different degrees of improvement compared to the model group. With the increase in drug administration time, the rats' breathing gradually stabilized, nasal secretions decreased, and their diet, hair loss, and mental state recovered to varying degrees. Figure 3As shown, the body weight of the model group decreased significantly compared with the blank group on the 7th day (***p<0.001); on the 14th day, the body weight of the rats in the pirfenidone group, composition group 1 and composition group 2 began to increase significantly compared with the model group (#p<0.05, ##p<0.01), and the trend was maintained until the end of the 28th day; on the 21st day, the body weight of the baicalin nanocrystal group increased significantly compared with the baicalin raw material group (▽p<0.05), and the trend was maintained until the end of the 28th day; on the 28th day, the body weight of the rats in the composition group 2 increased significantly compared with the baicalin nanocrystal group and the verbena extract group (▲p<0.05). The results show that after baicalin is made into nanocrystals, it can improve rat pulmonary fibrosis better than the raw material, and the composition composed of baicalin and verbena has more advantages than the two extracts administered alone, and can significantly improve the weight loss of rats caused by pulmonary fibrosis.

[0047] 4.3.2 Staining of rat lung tissue sections HE-stained and MASSON-stained sections of rat lung tissue were obtained and observed under an optical microscope.

[0048] HE staining results showed that the blank group ( Figure 2 A) The lung tissue capsule structure is clear, and the lung substance is the numerous alveoli at the ends of the bronchial branches at all levels in the lungs, without collagen fiber hyperplasia; model group ( Figure 2 B) in the figure shows flake-like alveolar consolidation, unclear alveolar structure, more 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 infiltration of lymphocytes around blood vessels and bronchioles (blue arrows); occasional perivascular edema (purple arrows), loosely arranged connective tissue, widened spaces, with a small amount of punctate infiltration of lymphocytes; 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, the lung substance is the numerous alveoli at the ends of the bronchial branches at all levels in the lungs, the alveolar wall is composed of a single layer of epithelium, focal alveolar wall capillary congestion (orange arrow), rare vascular congestion (orange arrow), a small amount of brown-yellow pigment deposition in the alveolar wall (brown arrow), showing mild lesions; Baicalin API group ( Figure 2 D) A small amount of granulocytes and mast cells 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); a large number of 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 2E) A small number of foam cells and mast cells in the alveoli (dark red arrows), occasional perivascular edema (purple arrows), loose connective tissue, 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 granulocytes, macrophages and mast cells can be seen in the alveolar wall and alveolar cavity (green arrows), moderate thickening of alveolar wall in a small area, widening of alveolar septa, a small amount of necrotic cell fragments in the alveoli (black arrows), rare vascular congestion (orange arrows), a small amount of brown-yellow pigment deposition (brown arrows), occasional bronchiolar epithelial hyperplasia (light green arrows), showing mild lesions; Composition 1 ( Figure 2 In group G, there was a small amount of granulocyte infiltration in the alveolar wall (green arrow), mild thickening of the alveolar wall in multiple foci, widening of the alveolar septum, a small amount of macrophages in the alveoli (dark red arrow), more alveolar expansion (dark blue arrow), irregular arrangement of bronchiolar epithelial cells (light green arrow), and a small amount of epithelial cells in the cavity, showing mild lesions; group 2 ( Figure 2 H) The lung tissue capsule structure is clear. The lung parenchyma 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. There are many foam cells in the alveoli (dark red arrows) and few vascular congestion (orange arrows), showing a mild lesion.

[0049] The results of MASSON staining showed that the blank group ( Figure 2 No obvious collagen fiber proliferation was observed in lung tissue; 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 Figure 1, a small amount of collagen fiber hyperplasia (yellow arrow) can be seen in the lung tissue. 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) can be seen in lung tissue. The collagen fibers are thin, discontinuous and irregularly arranged. 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 hyperplasia (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 fine.

[0050] The severity of lesions: model group > verbena extract group > baicalin raw material group > combination group 1 > baicalin nanocrystal group > positive drug group > combination group 2.

[0051] 4.3.3 MICRO-CT images of rat lungs It can be seen that the density of bilateral lung parenchyma in the blank group is uniform, the lung texture is clear and evenly distributed, and the bronchial bundles and vascular bundles can be seen gradually becoming thinner from the inside to the outside of the lungs, and the texture shadows formed by regular running are not found in the field of vision of both lungs; the lung parenchyma density in the model group increased, the texture in the lungs was blurred, large nodular shadows or mass shadows were seen, the lung structure was disordered, and the lung interstitial structure was abnormal; the other drug-treated groups showed different 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 > combination group 1 > positive drug group > combination group 2.

[0052] 4.3.4 Rat lung coefficient levels 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, compared with the blank group, the lung coefficient of the model group was significantly increased (n=10, ***p<0.001), and significantly decreased in the positive drug group, baicalin raw material group, baicalin nanocrystal group, combination group 1 and combination group 2 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 reduced (n=10, ▽p<0.05); compared with the baicalin nanocrystal group and the verbena extract group, the lung coefficient of the combination group 2 was significantly reduced (n=10, ▲p<0.05, △p<0.05).

[0053] 4.3.5 Total protein levels in rat lung tissue homogenates The total protein content in the bronchoalveolar lavage fluid of each group of rats was determined by BCA method. Figure 6 As shown, the total protein content of the blank group was (7.99±1.50) mg / mL, and the total protein content of 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 of the positive drug group, baicalin nanocrystal group, verbena extract group, composition 1 and composition 2 groups were significantly reduced (n=10, #p<0.05, ##p<0.01). Compared with the verbena extract group, the total protein content of the composition 2 group was significantly reduced (n=10, △p<0.05).

[0054] 4.3.6 Determination of IL-4, IL-6, IL-1β and TGF-β1 contents in rat lung tissue homogenate 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 7 As 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, combination group 1 and combination group 2 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 contents of IL-4, IL-6 and TGF-β1 in composition 2 were significantly reduced (n=10, ▲p<0.05, △p<0.05).

[0055] 4.3.7 Rat lung function level from Figure 8 It can be seen from the pulmonary function indicators that compared with the blank group, the lung function parameters of the model group rats Ti (inspiratory duration), Te (expiratory duration), PIF (maximum inspiratory flow), TV (tidal volume), MV (minute volume), RT (relaxation time) were significantly reduced, Penh (bronchial contraction parameter) and f (respiratory frequency) were significantly increased (n=10, ***p<0.001); the respiratory indicators of the drug-treated group were significantly improved compared with the model group (n=10, #p<0.05, ##p<0.01, ###p<0.001); except Te, the other lung function parameters of the baicalin nanocrystal group were significantly improved 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 the indicators of Ti, Te, PEF, MV, Penh and f (n=10, △p<0.05, ▲p<0.05, ▲▲p<0.01).

[0056] The above experimental results show that both baicalin and verbena can reduce the symptoms of pulmonary fibrosis in rats by reducing the content of inflammatory factors, total protein content and collagen deposition, and have a certain therapeutic effect on the treatment of pulmonary fibrosis. After baicalin is made into nanocrystals, its efficacy in various indicators of idiopathic pulmonary fibrosis is better than that of the raw material. 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. A Chinese medicine composition for treating idiopathic pulmonary fibrosis, characterized in that: Includes verbena extract and baicalin nanocrystals.

2. The Chinese medicine composition according to claim 1, characterized in that: The verbena extract is prepared according to the following method: taking verbena material, crushing it and passing it through a 40-mesh sieve, adding 10-12 times the amount of distilled water for reflux extraction, and extracting it twice, each time for 2 hours; after the extraction is completed, combining the extracts, concentrating under reduced pressure and freeze-drying to obtain the verbena extract.

3. The Chinese medicine composition according to claim 1 or 2, characterized in that: The total amount of verbenaside and verbenaside in the verbena extract is more than 3.03%.

4. The Chinese medicine composition according to claim 1 or 2, characterized in that: The content of baicalin in baicalin nanocrystals is 65%-85%.

5. The Chinese medicine composition according to claim 1, characterized in that: The baicalin nanocrystals are nanoparticles prepared by high-pressure homogenization method by placing baicalin in a poloxamer 188 aqueous solution with a mass percentage concentration of 0.1%-0.2%.

6. The Chinese medicine composition according to claim 5, characterized in that: The mass percentage concentration of the poloxamer 188 aqueous solution is 0.1%.

7. The Chinese medicine composition according to claim 5, characterized in that: The mass percentage concentration of the poloxamer 188 aqueous solution is 0.15%.

8. The Chinese medicine composition according to claim 5, characterized in that: The mass percentage concentration of the poloxamer 188 aqueous solution is 0.2%.

9. The Chinese medicine composition according to claim 5, 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) adding the pre-dispersed mixture into a high-pressure homogenizer, circulating it for 5 times at 300 bar, and then circulating it for 25 times at 1000 bar to obtain a nanocrystal suspension; (3) Freeze-drying the nanocrystal suspension to obtain baicalin nanocrystals.

10. The Chinese medicine composition according to claim 9, characterized in that: The particle size of the baicalin nanocrystal is 330-400nm.

11. The Chinese medicine composition according to any one of claims 1, 9 or 10, characterized in that: The mass ratio of the verbena extract to the baicalin nanocrystals is 1-5:0.5-2.

12. The Chinese medicine composition according to claim 11, characterized in that: The mass ratio of the verbena extract to the baicalin nanocrystals is 2:

1.

13. The Chinese medicine composition according to claim 11, characterized in that: The mass ratio of the verbena extract to the baicalin nanocrystals is 3:

1.

14. The Chinese medicine composition according to claim 11, characterized in that: The mass ratio of the verbena extract to the baicalin nanocrystals is 5:

2.

15. Use of the Chinese medicine composition according to any one of claims 1 to 14 in the preparation of a medicament for treating idiopathic pulmonary fibrosis.

16. The use according to claim 15, characterized in that: The Chinese medicine composition is prepared into an oral preparation.

17. The use according to claim 16, characterized in that: Oral preparations include tablets, granules, capsules, and oral solutions.

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