Application of small leaf ficus extract in preparation of medicine for treating emphysema

Abstract

The application discloses application of a small-leaf fig extract and component monomers vitexin and isovitexin of the small-leaf fig extract in preparation of a drug for relieving a lung emphysema process. The small-leaf fig extract and the component monomers vitexin and isovitexin of the small-leaf fig extract have the functions of targeting iron death treatment of epithelial cell death of lung emphysema. In a cell and an animal infection model, the small-leaf fig extract and the component monomers vitexin and isovitexin can inhibit peripheral blood eosinophil increase, restore lung function, reduce lung emphysema pathology, inhibit lung chronic inflammation and reduce lung epithelial barrier damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Ficus microcarpa extract in the preparation of drugs for treating emphysema. Background Technology

[0002] Emphysema is one of the main pathological manifestations of chronic obstructive pulmonary disease (COPD), characterized by alveolar wall structure destruction, decreased elastic recoil, and airflow obstruction. The pathogenesis of emphysema is complex, resulting from dynamic and cumulative environmental-gene-time interactions involving a complex network of various inflammatory cells and cytokines. These include: abnormal lung tissue development, protease / antiprotease imbalance, oxidant / antioxidant imbalance, and abnormal death of lung parenchymal cells.

[0003] Current clinical treatment for emphysema mainly relies on bronchodilators, inhaled corticosteroids, and oxygen therapy. However, these treatments have drawbacks such as long-term use, numerous side effects, drug resistance over time, and lack of response in some individuals. Therefore, finding safe and effective treatments remains a key focus of COPD research.

[0004] Recent studies suggest that abnormal death of lung epithelial cells is a core driver of emphysema progression. Partoptosis in lung epithelial cells leads to distal airway collapse and obstruction, alveolar structural damage, and alveolar fusion, exacerbating the over-expansion of emphysema. Partoptosis-induced epithelial cells release damage-related molecular patterns, activating the immune system and triggering a more intense inflammatory response, creating a vicious cycle that accelerates emphysema progression. Therefore, targeting key nodes in the partoptosis pathway has become a novel treatment strategy for emphysema.

[0005] Small-leaved Ficus ( Ficus microcarpa *Ficus microcarpa* (Lf) is a tree belonging to the genus *Ficus* in the family Moraceae. It is a traditional Chinese medicinal herb with anti-inflammatory, heat-clearing, cough-relieving, and phlegm-reducing effects. *Ficus microcarpa* extract is an extract from the dried leaves of *Ficus microcarpa*. Ficus microcarpa DEFM contains various chemical components such as flavonoids and phenolic acids. Among them, the flavonoid compounds vitexin (CAS No. 3681-93-4) and isovitexin (CAS No. 38953-85-4) have anti-inflammatory and antioxidant pharmacological activities. Currently, research on Ficus microcarpa extract and its monomeric components vitexin and isovitexin mainly focuses on improving asthma and acute lung injury. Its application in targeted ferroptosis therapy for emphysema has not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a drug that can effectively alleviate the progression of emphysema.

[0007] To achieve the above-mentioned objectives, this invention provides the application of Ficus microcarpa extract in the preparation of drugs for treating emphysema.

[0008] Preferably, the Ficus microcarpa extract is a Ficus microcarpa leaf extract.

[0009] Preferably, the Ficus microcarpa extract contains at least one of vitexin and isovitexin.

[0010] Preferably, the total content of vitexin and isovitexin in each 360mg of the Ficus microcarpa extract is not less than 0.28mg.

[0011] The present invention also provides the use of vitexin and / or isovitexin in the preparation of medicaments for treating emphysema.

[0012] Preferably, the drug is used to improve lung pathological damage and / or lung function in patients with emphysema.

[0013] Preferably, the pathological lung damage includes alveolar septal rupture and fusion, and alveolar cavity enlargement; the improvement of lung function includes reducing functional residual capacity, reducing string compliance, reducing inspiratory resistance, and increasing FEV1. 50 One or more of the following: FVC ratio, increase in maximal mid-expiratory flow, and restoration of dynamic lung compliance.

[0014] Preferably, the drug is used to suppress chronic inflammation of the lungs.

[0015] More preferably, the drug reduces one or more of the following: the total number of leukocytes and the proportion of eosinophils in peripheral blood; the levels of pro-inflammatory cytokines and matrix metalloproteinases in bronchoalveolar lavage fluid; and the total number of cells and the proportion of neutrophils in bronchoalveolar lavage fluid.

[0016] More preferably, the pro-inflammatory cytokine is selected from at least one of IL-6, TNF-α, and IL-1β.

[0017] More preferably, the matrix metalloproteinase is selected from at least one of MMP-9 and MMP-2.

[0018] Preferably, the drug is used to protect the lung barrier function.

[0019] More preferably, the drug upregulates the expression of the epithelial marker E-cadherin and / or the tight junction protein ZO-1 in lung tissue.

[0020] Preferably, the drug is used to inhibit ferroptosis in lung epithelial cells.

[0021] More preferably, the drug upregulates the expression of GPX4, SLC7A11, SLC40A1 and / or FTL proteins, downregulates the expression of ACSL4 and / or TFR proteins, and reduces the levels of lipid peroxidation products and / or ferrous ions, or one or more of these.

[0022] This invention utilizes porcine pancreatic elastase (PPE) administered intratracheally to establish a mouse model of emphysema. The modeling period is 4 weeks, and the aforementioned drug is administered starting in the 3rd week of modeling, with continuous administration for 2 weeks. This treatment regimen verifies the interventional effects of *Ficus microcarpa* extract and its components vitexin and isovitexin on existing emphysema damage. Results show that treatment with *Ficus microcarpa* extract and its components vitexin and isovitexin can inhibit the increase of peripheral blood eosinophils, restore lung function, reduce emphysema pathology, suppress chronic lung inflammation, and reduce damage to the pulmonary epithelial barrier, thereby alleviating the progression of emphysema. Attached Figure Description

[0023] Figure 1 This study demonstrates the effects of Ficus microcarpa extract and its constituent monomers vitexin and isovitexin on improving lung pathology and function in mice with emphysema after treatment. (a) H&E staining of lung tissue and statistical analysis of alveolar spacing; (b) Lung function tests, including FRC (functional residual capacity), Cchord (string compliance), RI (inspiratory resistance), and FEV1. 50 / FVC (forced expiratory volume to forced vital capacity at 50 ms), MMEF (maximum mid-expiratory flow), and Cdyn (dynamic lung compliance).

[0024] Figure 2 This study demonstrates the inhibitory effects of Ficus microcarpa extract and its constituent monomers vitexin and isovitexin on chronic lung inflammation. (a) Blood routine indicators; (b) ELISA detection of inflammatory markers in bronchoalveolar lavage fluid; (c) Flow cytometry detection of inflammatory cell infiltration and statistical proportion in bronchoalveolar lavage fluid.

[0025] Figure 3 This study demonstrates that the extract of *Ficus microcarpa* and its constituent monomers vitexin and isovitexin protect the lung barrier. Immunofluorescence staining results of lung tissue, including E-cadherin (epithelial cells) and ZO-1 (tight linker protein), are also shown.

[0026] Figure 4 The results showed that the extract of Ficus microcarpa and its components vitexin and isovitexin significantly inhibited ferroptosis in lung epithelial cells. (a) Western blot analysis of target protein expression levels; (b) statistical analysis of relative changes in target bands in Western blot analysis; (c) detection of ferroptosis markers: lipid peroxidation and ferrous ions. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0030] The Ficus microcarpa extract (also known as Ficus microcarpa dry extract, an extract from the dried leaves of the Ficus microcarpa plant, belonging to the Moraceae family) is a commercially available product, purchased from Zhongshan Zhongzhi Pharmaceutical Group Co., Ltd., batch number 2412052A. According to page 1275 of Part I of the 2020 edition of the Chinese Pharmacopoeia, the total content of vitexin and isovitexin in each 360mg Ficus microcarpa extract shall not be less than 0.28mg.

[0031] Establishment of the PPE emphysema mouse model: C57BL / 6 mice (8-12 weeks old, weighing 20-25 g) were anesthetized with gas and 0.6 U / 50 μL of porcine pancreatic elastase (MCE, HY-P2974) was instilled into each nasal cavity once a week for four consecutive weeks. The groups included a normal control group, a model group, a positive control group (Roflumilast: 5 mg / kg / d), a low-dose vitexin group (Vitexin-L: 30 mg / kg / d), a high-dose vitexin group (Vitexin-H: 90 mg / kg / d), a low-dose isovitexin-L group (30 mg / kg / d), a high-dose isovitexin-H group (90 mg / kg / d), and a Ficus microcarpa extract group (DEFM: 1.944 g / kg / d). After the third nasal instillation, the mice were administered the drug via gavage once daily for 14 consecutive days. Lung function (functional residual capacity, forced expiratory volume to forced vital capacity at 50 ms, and forced expiratory volume to forced vital capacity) of mice were assessed 28 days after the first nasal instillation. 50 / FVC, mean mid-expiratory flow rate (MMEF), inspiratory resistance (RI), string compliance (Cchord), dynamic lung compliance (Cdyn), peripheral blood routine blood routine test for eosinophil percentage, lung HE pathology alveolar linear intercept, BALF sample for macrophage / neutrophil ratio and inflammatory damage factors such as IL-6, TNF-α, IL-1β, MMP-2, and MMP-9, and immunofluorescence detection of intercellular junctions (ZO-1, E-cadherin) in lung tissue.

[0032] Example 1. Effects of Ficus microcarpa extract and its components vitexin and isovitexin on improving lung pathology and function in mice with emphysema after treatment. The results are as follows Figure 1 As shown in the figure, the results indicate that the extract of Ficus microcarpa and its active monomers vitexin and isovitexin significantly improve the pathological damage to lung tissue and multiple lung function indicators in mice with PPE-induced emphysema.

[0033] In terms of lung pathology, lung tissues from mice in each group were collected for pathological sections and H&E staining. The results showed that mice in the PPE model group exhibited obvious alveolar septal breakage and fusion, and significantly enlarged alveolar cavities, presenting typical emphysema changes. However, after intervention in each drug-treated group, the degree of alveolar structural damage was reduced, alveolar dilation was alleviated, and septal fusion was decreased. Figure 1 ,a).

[0034] In terms of lung function assessment, compared with the model group, the functional residual capacity (FRC) of mice in the drug-treated group was reduced, indicating that lung hyperinflation and increased static lung volume were alleviated; chord compliance (Cchord) decreased, reflecting the correction of abnormally elevated lung compliance; inspiratory resistance (RI) decreased, indicating improved airway resistance; the FEV50 / FVC ratio increased and the maximum mid-expiratory flow (MMEF) increased, indicating that airway obstruction and expiratory flow limitation were reduced, and small airway function was also restored; at the same time, dynamic lung compliance (Cdyn) tended to normalize, comprehensively reflecting that the abnormal state of decreased lung elastic recoil and increased airway resistance was effectively reversed. Figure 1 b).

[0035] In summary, the extract of Ficus microcarpa and its monomeric components can improve lung lesions in mice with emphysema at both the pathological and functional levels.

[0036] Example 2. The inhibitory effect of Ficus microcarpa extract and its components vitexin and isovitexin on chronic lung inflammation. Emphysema is a long-term chronic inflammatory disease, often accompanied by a chronic inflammatory phenotype.

[0037] like Figure 2As shown, the inhibitory effect of the drug of the present invention on chronic lung inflammation was also observed, including in peripheral blood and local lung areas. In blood routine tests, compared with the normal control group, the total white blood cell count and the proportion of eosinophils in the peripheral blood of mice in the PPE model group were significantly increased, while after administration of Ficus microcarpa extract and its monomers vitexin and isovitexin, the above indicators were significantly inhibited (e.g., Figure 2 ,a).

[0038] Simultaneously, ELISA was used to detect inflammatory factors in bronchoalveolar lavage fluid (BALF). The results showed that each treatment group significantly reduced the expression levels of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β, as well as matrix metalloproteinases MMP-9 and MMP-2, in the BALF of emphysematous mice, indicating that the drugs could effectively alleviate lung inflammatory damage (such as...). Figure 2 b).

[0039] Furthermore, flow cytometry analysis of cellular components in BALF revealed that both the total number of cells and the proportion of neutrophils in BALF significantly decreased after drug treatment (e.g., Figure 2 (c) further confirmed that the extract and its monomeric components have a significant inhibitory effect on emphysema-related chronic inflammation.

[0040] Example 3. Protective effect of Ficus microcarpa extract and its constituent monomers vitexin and isovitexin on the lung barrier. like Figure 3 Immunofluorescence staining results showed that the expression levels of epithelial markers E-cadherin and tight junction protein ZO-1 in the lung tissue of mice in the emphysema model group were significantly reduced, indicating that the integrity of the lung epithelial barrier was impaired.

[0041] After intervention with Ficus microcarpa extract and its active monomers vitexin and isovitexin, the fluorescence intensity of the two proteins was significantly enhanced, and their expression levels returned to near normal, indicating that the drug can effectively reverse the reduction of epithelial junction proteins caused by emphysema and play a role in protecting the lung barrier function.

[0042] Example 4. The extract of Ficus microcarpa and its constituent monomers vitexin and isovitexin significantly inhibited ferroptosis in lung epithelial cells. To further investigate the effect of the drug on ferroptosis in lung epithelial cells, ferroptosis markers were detected.

[0043] like Figure 4 The results showed that lung tissue in the PPE model group exhibited typical ferroptosis-related molecular changes: the expression levels of ferroptosis core defense protein GPX4, antioxidant-related protein SLC7A11, iron efflux protein SLC40A1, and iron storage protein FTL were significantly decreased, while the expression of lipid peroxidase ACSL4 and iron uptake-related protein TFR were significantly increased; simultaneously, lipid peroxidation products and ferrous ions (Fe²⁺) were also significantly increased.+ The levels of these proteins were also significantly increased in the model group. After intervention with Ficus microcarpa extract and its monomers vitexin and isovitexin, the abnormal expression of the above proteins was significantly reversed; GPX4, SLC7A11, SLC40A1, and FTL increased, while ACSL4 and TFR decreased. Figure 4 (a, b), and lipid peroxidation and Fe² + The level also declined significantly. Figure 4 (c) indicates that the extract and its active monomers can effectively inhibit emphysema-induced ferroptosis in lung epithelial cells, thereby exerting a cytoprotective effect.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Application of Ficus microcarpa extract in the preparation of drugs for treating emphysema.

2. The application according to claim 1, characterized in that, The Ficus microcarpa extract is an extract of Ficus microcarpa leaves.

3. The application according to claim 1, characterized in that, The Ficus microcarpa extract contains at least one of vitexin and isovitexin.

4. The use of vitexin and / or isovitexin in the preparation of drugs for treating emphysema.

5. The application according to any one of claims 1 to 4, characterized in that, The drug is used to improve lung pathological damage and / or lung function in patients with emphysema.

6. The application according to claim 5, characterized in that, The lung pathological damage includes alveolar septal breakage and fusion, and alveolar cavity enlargement; the improvement of lung function includes reducing functional residual capacity, reducing string compliance, reducing inspiratory resistance, and increasing FEV1. 50 One or more of the following: FVC ratio, increase in maximal mid-expiratory flow, and restoration of dynamic lung compliance.

7. The application according to any one of claims 1 to 4, characterized in that, The drug is used to suppress chronic inflammation in the lungs.

8. The application according to claim 7, characterized in that, The drug reduces one or more of the following: the total white blood cell count and eosinophil percentage in peripheral blood; the levels of pro-inflammatory cytokines and matrix metalloproteinases in bronchoalveolar lavage fluid; and the total cell count and neutrophil percentage in bronchoalveolar lavage fluid. Preferably, the pro-inflammatory cytokines are selected from at least one of IL-6, TNF-α, and IL-1β. Preferably, the matrix metalloproteinases are selected from at least one of MMP-9 and MMP-2.

9. The application according to any one of claims 1 to 4, characterized in that, The drug is used to protect lung barrier function, preferably by upregulating the expression of the epithelial marker E-cadherin and / or tight junction protein ZO-1 in lung tissue.

10. The application according to any one of claims 1 to 4, characterized in that, The drug is used to inhibit ferroptosis in lung epithelial cells. Preferably, the drug upregulates the expression of GPX4, SLC7A11, SLC40A1 and / or FTL proteins, downregulates the expression of ACSL4 and / or TFR proteins, and reduces the levels of lipid peroxidation products and / or ferrous ions, or one or more of these.

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