Use of volatile oil from artemisia rupestris l. in preparation of medicine for preventing or treating pulmonary fibrosis

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Artemisia capillaris volatile oil in the preparation of drugs for the prevention or treatment of pulmonary fibrosis. This invention uses a bleomycin (BLM)-induced mouse pulmonary fibrosis model to explore the therapeutic effect of Artemisia capillaris volatile oil on pulmonary fibrosis. The effects of Artemisia capillaris volatile oil on the pulmonary fibrosis model were detected by methods such as tissue H&E staining and MASSON staining. The results show that, during the course of pulmonary fibrosis in mice, Artemisia capillaris volatile oil can reduce the degree of lung tissue structural damage, alleviate the degree of inflammatory cell infiltration, reduce pulmonary fibrosis changes, alleviate collagen fiber deposition, improve the survival rate of mice with pulmonary fibrosis, and reduce hydroxyproline content, effectively alleviating the degree of BLM-induced pulmonary fibrosis in mice. Artemisia capillaris, as a traditional Chinese medicine, has advantages such as being natural, economical, and easily accessible, and can provide an effective treatment option for patients with pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Artemisia capillaris volatile oil in the preparation of drugs for the prevention or treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a chronic, irreversible, progressive fibrotic lung disease. It is the end-stage pathological change of various interstitial lung diseases (ILDs). It is characterized by abnormal remodeling of lung tissue, accompanied by continuous fibroblast activation and massive extracellular matrix (ECM) deposition, leading to thickening of lung septa and impaired gas exchange, ultimately causing respiratory failure.

[0003] Many types of interstitial lung diseases (ILDs) can develop into pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is the most common fibrotic disease. Other ILDs include exposure-related ILDs (such as silicosis), drug-induced pulmonary fibrosis, autoimmune-related ILDs (such as rheumatoid arthritis and systemic sclerosis), and idiopathic nonspecific interstitial pneumonia. Patients with pulmonary fibrosis often present with progressive dyspnea, dry cough, and shortness of breath on exertion, eventually leading to respiratory failure and even death. With advancements in diagnostic technology and an aging population, the incidence and mortality rates of pulmonary fibrosis continue to rise.

[0004] The pathogenesis of pulmonary fibrosis is complex, involving interactions among various cells, such as epithelial cells, endothelial cells, macrophages, and fibroblasts. Among these, fibroblast activation and their differentiation into myofibroblasts are core components of pulmonary fibrosis progression. Pulmonary fibrosis begins with repeatedly damaged epithelial cells, leading to the release of pro-inflammatory factors and pro-fibrotic mediators, which in turn activate fibroblasts and promote extracellular matrix (ECM) deposition. Excessive ECM deposition further increases tissue stiffness, creating a vicious cycle that leads to the continuous progression of pulmonary fibrosis. Although considerable research has been conducted on the regulatory mechanisms of fibroblast activation and ECM deposition, the complex interactions involving multiple signaling pathways and cell types in pulmonary fibrosis present significant challenges in developing drugs that can precisely intervene in key aspects with minimal side effects.

[0005] Currently, there are two FDA-approved drugs for the treatment of pulmonary fibrosis: pirfenidone (PFD) and nintedanib. These two drugs primarily work by inhibiting fibroblast proliferation and ECM deposition to slow disease progression, but their efficacy is limited, and they have significant side effects and high treatment costs. Specifically, pirfenidone reduces fibrosis by inhibiting the TGF-β signaling pathway, but its efficacy is weak, and common side effects include gastrointestinal reactions, photosensitivity, and abnormal liver function. Nintedanib, as a multi-target tyrosine kinase inhibitor, can inhibit fibroblast proliferation and angiogenesis, but its side effects include diarrhea, nausea, and hepatotoxicity, and it is expensive.

[0006] Tibetan medicinal artemisia is Artemisia capillaris, a plant in the Asteraceae family. Artemisia subdigitata The dried aerial parts of *Artemisia argyi* are one of the sources of Tibetan medicine, specifically the *Pulmang* type, and possess antitussive, expectorant, anti-inflammatory, antibacterial, antimalarial, antitumor, and antioxidant properties. Currently, there are no reports on the activity of *Artemisia argyi* volatile oil in treating pulmonary fibrosis. Exploring the efficacy and mechanism of *Artemisia argyi* volatile oil in treating pulmonary fibrosis is of great significance for developing a drug with significant efficacy, few side effects, and low cost for the treatment of pulmonary fibrosis. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and to provide the application of Artemisia capillaris volatile oil in the preparation of drugs for the prevention or treatment of pulmonary fibrosis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of Artemisia capillaris volatile oil in the preparation of drugs for the prevention or treatment of pulmonary fibrosis.

[0009] Alternatively, in the above applications, the pulmonary fibrosis is either idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

[0010] For example, the pulmonary fibrosis is bleomycin-induced pulmonary fibrosis.

[0011] As an alternative approach, in the above applications, the volatile oil from Artemisia capillaris is said to alleviate the progression of pulmonary fibrosis and reduce mortality during the pulmonary fibrosis process.

[0012] As an alternative approach, in the above applications, the volatile oil from Artemisia capillaris is characterized by reducing the degree of damage to lung tissue structure, alleviating the degree of inflammatory cell infiltration, and reducing pulmonary fibrosis changes during the process of pulmonary fibrosis.

[0013] Alternatively, in the above applications, the volatile oil from Artemisia capillaris is said to reduce hydroxyproline content during pulmonary fibrosis.

[0014] Alternatively, in the above applications, the Artemisia capillaris volatile oil is used as the sole active ingredient to prevent or treat pulmonary fibrosis.

[0015] Alternatively, in the above applications, the medicament may include other drugs clinically used to treat pulmonary fibrosis.

[0016] Preferably, the other drugs used clinically to treat pulmonary fibrosis are pirfenidone or nintedanib.

[0017] Alternatively, in the above applications, the drug may also contain pharmaceutically acceptable excipients.

[0018] Alternatively, in the above applications, the dosage form of the drug is tablets, capsules, granules, powders, oral liquids, or injections.

[0019] Alternatively, in the above applications, the Artemisia capillaris volatile oil is Artemisia capillaris oil soft capsules.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the therapeutic effect of Artemisia capillaris volatile oil on pulmonary fibrosis. The effects of Artemisia capillaris volatile oil on the pulmonary fibrosis model were detected using methods such as tissue H&E staining and MASSON staining. The results showed that Artemisia capillaris volatile oil, during the course of pulmonary fibrosis in mice, could reduce the degree of lung tissue structural damage, alleviate the degree of inflammatory cell infiltration, reduce pulmonary fibrosis changes, alleviate collagen fiber deposition, improve the survival rate of mice with pulmonary fibrosis, and reduce hydroxyproline content, effectively alleviating the degree of BLM-induced pulmonary fibrosis in mice. As a naturally derived drug, Artemisia capillaris volatile oil has advantages such as being natural, economical, and easily accessible, and can provide an effective treatment option for patients with pulmonary fibrosis. Attached Figure Description

[0021] Figure 1 Figure A shows the effect of NWH on the body weight of mice with BLM-induced pulmonary fibrosis, and Figure B shows the survival curves of mice with pulmonary fibrosis in each group. Figure 2 The lung tissue morphology of mice in the BLM group; Figure 3 Morphology of lung tissue in PFD group mice; Figure 4 The lung tissue morphology of NWHH group mice; Figure 5 The lung tissue morphology of NWHM group mice; Figure 6 H&E staining results (×200); Figure 7MASSON staining results (×200); Figure 8 The results are from histopathological examination. Figure A shows the alveolitis score; Figure B shows the relative collagen volume results. Figure 9 This is a graph showing the changes in hydroxyproline in lung tissue of each group. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The following are the experimental methods and results used in the implementation examples.

[0023] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0025] Example: Pharmacodynamic Study Trial 1. Laboratory animals and materials SPF-grade male C57BL / 6J mice (6-8 weeks old, 22-24 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The animals were housed at the Animal Experiment Center of Shenyang Pharmaceutical University under 12-hour light, constant temperature of 24℃, and free access to food and water.

[0026] Pirfenidone (Beijing Contini Pharmaceutical Co., Ltd.; Product Batch No. 20220705; Approval Number: National Drug Approval Number H20133376); Bleomycin for Injection (Hanhui Pharmaceutical Co., Ltd.; Product Batch No. 22037911; Approval Number: National Drug Approval Number H20055883); Hydroxyproline Assay Kit was purchased from Nanjing Jiancheng Bioengineering Technology Co., Ltd.

[0027] 2. Preparation of experimental reagents Bleomycin: Dissolve bleomycin (BLM) in physiological saline to obtain a 4 mg / kg BLM working solution. Prepare immediately before use.

[0028] Preparation of anesthetic: Add 1 g of tribromoethanol powder to 630 μL of tert-amyl alcohol, shake well until the tribromoethanol dissolves, and prepare aphrodine stock solution. Store the stock solution at room temperature, protected from light. Dilute the stock solution with 0.9% physiological saline to 2.5% aphrodine and store at 4°C, protected from light.

[0029] 3. Establishment of a mouse model of pulmonary fibrosis, animal grouping, and drug administration. In this study, a mouse pulmonary fibrosis model was established by inducing bleomycin at 4 mg / kg. The mice were treated with different doses of Artemisia capillaris volatile oil (NWH) the day after modeling. After 21 days of administration, the lung tissue of the mice was collected for subsequent experiments.

[0030] After 1 day of acclimatization, C57BL / 6J mice were used to induce a pulmonary fibrosis model using bleomycin. For 12 hours prior to modeling, mice were fasted but allowed free access to water. Surgical instruments were sterilized and dried using high-temperature steam. Mice were weighed preoperatively and anesthetized intraperitoneally with 2.5% aphthylamine (250 mg / kg). They were then fixed to a rack. The mouse's tongue was pulled forward and to the left with forceps, and alternating light and dark areas of the glottis were visible under illumination. A 22 G cannula was inserted into the trachea through the glottis, and 4 mg / kg BLM working solution was injected into the bottom of the cannula. The mice were allowed to freely inhale BLM through the trachea. They were then rotated three times to ensure even diffusion of the drug in the lungs. After standing upright for 5 minutes, they were placed on a warming pad to await recovery and then separated into different cages / groups.

[0031] In the experimental examples of this invention, Artemisia capillaris oil soft capsules (purchased from Qinghai Lukang Dadi Pharmaceutical Co., Ltd., product batch number 20231004, product specification: 0.2 g per capsule (containing 100 mg of Artemisia capillaris volatile oil)) were used. According to the instructions, the adult oral administration was 1 capsule three times a day, with a clinical dose of 5 mg / kg / day and a minimum dose of 45 mg / kg / day for mice. The dosage was measured based on the amount of Artemisia capillaris volatile oil.

[0032] The day after modeling, mice were randomly divided into four groups of 10 mice each: a positive control group (pirfenidone, PFD), a model control group (bleomycin, BLM), a low-dose Artemisia capillaris volatile oil group (NWHM), and a high-dose Artemisia capillaris volatile oil group (NWHH). The BLM group was given an equal volume of physiological saline by gavage, while the PFD group received 160 mg / kg daily. -1 Pirfenidone was administered by gavage, with the NWHM group receiving 90 mg / kg daily. -1 NWH (based on the volatile oil content of Artemisia capillaris in Artemisia capillaris oil soft capsules) was administered by gavage. The NWHH group received 180 mg / kg daily. -1 NWH (based on the volatile oil content of Artemisia capillaris in Artemisia capillaris oil soft capsules) was administered by gavage, as detailed in Table 1. Administration continued for 21 days, during which time all animals had unrestricted access to water and food. Daily recordings of mouse activity, general condition, weight, and survival status were made, as detailed in Tables 2 and 3. Figure 1 As shown.

[0033] Table 1. Animal grouping and drug administration (n=10) 4. Source materials Twelve hours after the last administration, mice were weighed, anesthetized with 2.5% aphthylamine, and euthanized after blood collection from the eyeballs. The mice were centrifuged at 3500 r / min for 15 min, and serum was collected for ELISA testing. Mice were locally disinfected with 75% ethanol, and the thoracic cavity was cut along the anterior midline to expose the trachea and heart and lungs. The trachea was ligated with surgical sutures, and 0.5 mL of PBS buffer solution was instilled once. After instillation, the thoracic cavity was kneaded, and bronchoalveolar lavage fluid was aspirated and collected. This process was repeated three times. The entire lungs were rinsed with physiological saline, photographed, dried with filter paper, and weighed. Figures 2-5 As shown in Table 3. The upper lobe of the right lung was cut off, and the left lung was fixed with 4% paraformaldehyde fixative. The remaining lung was stored at -80℃ for subsequent experiments. The upper lobe of the right lung was placed in a 2 mL EP tube and dried at 80℃ until constant weight. It was weighed, and the wet-to-dry weight ratio of the lung was calculated to reflect the degree of lung tissue edema.

[0034] 5. Histopathological examination Fixed lung tissue was dehydrated with graded ethanol, cleared, embedded in paraffin, sectioned (4 μm thick), dewaxed to water, stained with H&E and Masson staining, dehydrated, mounted with neutral glue, and the pathological changes of the lung tissue were observed under a microscope, as shown below. Figure 6 and Figure 7 As shown.

[0035] The Szapiel scoring system was used to evaluate the inflammation status of lung tissue by H&E staining, and the degree of inflammatory infiltration was graded from 0 to 3. The results are shown in Table 4 below. Figure 8 As shown in Figure A. Nine non-overlapping fields of view on the slide were observed and scored using a three-blind microscope. Five non-overlapping fields of view on the slide were randomly selected for imaging, and the relative collagen area was calculated using ImageJ. The results are shown in Table 5 below. Figure 8 As shown in B.

[0036] 6. Determination of hydroxyproline content The right residual lung of each group of mice was used for alkaline hydrolysis. According to the kit instructions, the tissue was placed in a test tube and 0.2 mL of hydrolysis solution was added. The mixture was hydrolyzed in a boiling water bath for 20 min. 2 μL of indicator was added, and the pH was adjusted to approximately 6.0. Distilled water was added to a final volume of 2 mL and mixed well. 1.5 mL of the hydrolysis solution was taken, and 10 mg of activated charcoal was added. The mixture was centrifuged at 3500 r / min for 10 min. The supernatant was collected, and the absorbance (A) was measured at 550 nm according to the instructions. See Table 6 below. Figure 9 As shown.

[0037] 7. Statistical Analysis Data were processed using Excel 16.88 and IBM SPSS 29.0.1.0. Multiple datasets were processed using Two-Way ANOVA, and single datasets were processed using One-Way ANOVA. For datasets with three or more datasets, significance was tested using ANOVA, and pairwise comparisons were performed using Tukey's multiple comparison method. P A value <0.05 indicates a statistically significant difference between groups. GraphPad Prism 8.0 software was used to create the statistical graph.

[0038] 8. Results and Analysis 8.1 Effects of NWH on body weight, quality of life, and survival rate in mice with BLM-induced pulmonary fibrosis Daily records were kept of the activity and general condition of mice in each group, as well as their weight and survival status, as detailed in Table 2 and [Table data missing]. Figure 1 As shown.

[0039] Table 2. Body weight records of mice in each group (Mean ± SD, n=7) Note: Compared with the BLM group, P <0.05, P <0.01, P <0.001; compared with the PFD group, # P <0.05, ## P <0.01, ### P <0.001.

[0040] From Table 2 and Figure 1 It was found that within three days after BLM inhalation modeling via the airway, the body weight of mice in all groups showed a decreasing trend. Mice exhibited lethargy, sluggish movement, rapid breathing, reduced food intake, and a significant decline in quality of life. Four days after modeling, the BLM group showed a significant decrease in body weight and the appearance of rales. Compared with the BLM group, as shown in Table 2, from day 3 to day 10 after modeling, the body weight of the NWHH group was significantly higher than that of the BLM group. P <0.05, P <0.01), and as time progressed, the weight difference among the groups of mice gradually decreased, and there was no significant difference in weight among the groups at the observation endpoint. However, the PFD group mice showed reduced food intake and a slower weight gain trend after 7 days of administration, which is presumably related to the effect of PFD on gastrointestinal function.

[0041] Four mice in the BLM group died between days 10 and 14 after modeling, one mouse in the PFD group died on day 14, and no mice died in the NWHH and NWHM groups. This indicates that administration of NWH during bleomycin-induced pulmonary fibrosis can significantly alleviate the progression of pulmonary fibrosis in mice and reduce the mortality rate. Since there was data from one BLM mouse in the preliminary experiment, n=7 was selected for subsequent calculations.

[0042] 8.2 Effects of NWH on lung index, pulmonary edema, and lung tissue morphology in BLM-induced pulmonary fibrosis mice Table 3. Lung coefficients, water content in the right upper lobe, and wet / dry weight ratio of the right upper lobe for each group (Mean ± SEM, n=7) Note: Compared with the BLM group, P <0.05, P <0.01, P <0.001; compared with the PFD group, # P <0.05, ## P <0.01, ### P <0.001.

[0043] Depend on Figures 2-5 As shown in Table 3, the visible changes in the appearance of the mouse lungs revealed varying degrees of peripulmonary bleaching, surface wrinkling, and petechiae in all groups. The experimental results indicate that NWH improved bleomycin-induced pulmonary edema in mice to some extent, but there was no significant difference between the groups.

[0044] Table 4 Szapiel scores of H&E stained sections (Mean±SEM, n=3) Note: Compared with the BLM group, P <0.05, P <0.01, P <0.001; compared with the PFD group, # P <0.05, ## P<0.01, ### P <0.001.

[0045] From Table 4, Figure 8 A and Figure 6 H&E staining results showed that, compared with the BLM group, the NWH treatment group mice had more intact lung tissue structure, less inflammatory cell infiltration, and reduced pulmonary fibrosis. Szapiel scoring (Table 4) of the H&E-stained sections assessed the degree of inflammation. Compared with the BLM group, the NWHH group mice showed a significantly reduced degree of inflammatory cell infiltration in lung tissue. P <0.01, the degree of inflammatory cell infiltration in the lung tissue of mice in the NWHM group was significantly reduced ( P The value <0.001 indicates that NWH has a significant effect on improving BLM-induced pulmonary fibrosis in mice.

[0046] Depend on Figure 6 H&E staining revealed significant thickening of the alveolar walls and a marked increase in exudate within the alveolar cavities in the BLM group mice. Extensive inflammatory cell infiltration was observed in the alveolar cavities and pulmonary interstitium, along with abundant fibroblast proliferation and fibroblast foci. The normal alveolar structure disappeared, with extensive lymphocyte invasion and collagen proliferation surrounding the trachea and alveoli, leading to the disappearance or adhesion of normal alveolar structure. The alveolar septa were significantly widened and thickened, and fibrous tissue was distributed in patches or cords. While alveolar walls were also thickened in the PFD, NWHH, and NWHM groups, the degree of inflammatory cell infiltration and fibrosis was reduced.

[0047] Table 5. Percentage of collagen fiber area in MASSON stained sections (Mean±SEM, n=5) Note: Compared with the BLM group, P <0.05, P <0.01, P <0.001; compared with the PFD group, # P <0.05, ## P <0.01, ### P<0.001.

[0048] From Table 5, Figure 8 B. Figure 7MASSON staining revealed that the BLM group showed more blue fibrous strands and collagen deposits in the alveoli, indicating alveolar structure disruption. Compared to the BLM group, the PFD and NWHM groups showed significantly reduced lung tissue structural damage. P <0.05), collagen fiber deposition is reduced.

[0049] 8.3 Effect of NWH on hydroxyproline content in lung tissue of mice with BLM-induced pulmonary fibrosis Table 6 Hydroxyproline content in lung tissue of mice in each group (Mean ± SEM, n=7) Note: Compared with the BLM group, P <0.05, P <0.01, P <0.001; compared with the PFD group, # P <0.05, ## P <0.01, ### P <0.001.

[0050] An elevated hydroxyproline content indicates a deeper degree of tissue fibrosis. (From Table 6 and...) Figure 9 It was found that, compared with the BLM group, the hydroxyproline content in the lung tissue of mice in both the PFD group and the NWHH group was significantly decreased. P <0.05), indicating that administration of 180 mg / kg / d NWH can effectively alleviate the degree of tissue fibrosis in mice with pulmonary fibrosis.

[0051] The above experiments demonstrate that NWH can alleviate the inflammatory response and collagen fiber deposition in the lung tissue of mice induced by BLM during the course of pulmonary fibrosis, improve the survival rate of mice with pulmonary fibrosis, and improve BLM-induced pulmonary fibrosis in mice. Artemisia capillaris volatile oil, as a naturally sourced medicine, has advantages such as being natural, economical, and readily available, and can provide an effective treatment option for patients with pulmonary fibrosis.

Claims

1. Application of Artemisia capillaris volatile oil in the preparation of drugs for the prevention or treatment of pulmonary fibrosis.

2. The application as described in claim 1, characterized in that, The pulmonary fibrosis is either idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

3. The application as described in claim 1 or claim 2, characterized in that, The volatile oil from Artemisia capillaris is said to alleviate the progression of pulmonary fibrosis and reduce mortality during the pulmonary fibrosis process.

4. The application as described in claim 3, characterized in that, The volatile oil from Artemisia capillaris is said to reduce the degree of damage to lung tissue structure, lessen the degree of inflammatory cell infiltration, and reduce pulmonary fibrosis changes during the process of pulmonary fibrosis.

5. The application as described in claim 1 or claim 2, characterized in that, The effect of the volatile oil from Artemisia capillaris is to reduce hydroxyproline content during pulmonary fibrosis.

6. The application as described in claim 1 or claim 2, characterized in that, The volatile oil of Artemisia capillaris is used as the sole active ingredient to prevent or treat pulmonary fibrosis.

7. The application as described in any one of claims 1 to 6, characterized in that, The medication includes other drugs clinically used to treat pulmonary fibrosis.

8. The application as described in claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application as described in claim 8, characterized in that, The dosage form of the drug is tablets, capsules, granules, powders, oral liquids, or injections.

10. The application as described in claim 1, characterized in that, The Artemisia capillaris volatile oil is an Artemisia capillaris oil soft capsule.

Images