Application of effective component combination of chrysanthemum indicum l. essential oil in preparation of drugs for preventing and treating acute lung injury

Abstract

The application discloses application of a wild chrysanthemum essential oil effective component combination in preparation of a medicine for preventing and treating acute lung injury, and particularly relates to the technical field of biological medicines, wherein the wild chrysanthemum essential oil effective component comprises beta-farnesene, 4-terpenol, artemisia oil, wild chrysanthemum alcohol and carvomenthone, and in an acute lung injury model, the wild chrysanthemum essential oil effective component can significantly regulate the survival state of lung epithelial cells, improve the phenomenon of cell activity decline, and effectively maintain the stability of mitochondrial membrane potential (Delta Psi m). In an acute lung injury mouse model, the wild chrysanthemum essential oil effective component combination can significantly improve the pathological injury of lung tissue, and significantly inhibit the expression of inflammatory mediators in bronchoalveolar lavage fluid (BALF). In summary, the wild chrysanthemum essential oil effective component combination can be applied in preparation of an anti-acute lung injury medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the combination of effective components of wild chrysanthemum essential oil in the preparation of drugs for the prevention and treatment of acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a common critical illness caused by a variety of direct or indirect factors, including sepsis, pneumonia, trauma, acute pancreatitis, and aspiration of gastric contents. Sepsis is the most common cause of ALI. The main pathophysiological features of ALI are diffuse alveolar damage, pulmonary edema, and excessive inflammatory response. Its clinical features are mainly decreased lung compliance, hypoxemia, and dyspnea, which can develop into acute respiratory distress syndrome (ARDS) in severe cases.

[0003] Modern medicine has not yet determined a definitive treatment for inflammation-related lung diseases. Treatment typically involves anti-inflammatory drugs to relieve symptoms and reduce lung damage. Nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and bronchodilators are commonly used to control the symptoms of inflammatory respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Lung-protective ventilation, fluid therapy, and neuromuscular blocking agents have shown some efficacy in the clinical treatment of ALI, but the mortality rate of ALI remains as high as 40%, with death often caused by multi-system organ failure due to inflammatory mediators. However, these drugs do not stop disease progression, and side effects are also a concern. For example, in respiratory infections caused by bacteria and viruses, the use of antibiotics can lead to infections caused by drug-resistant or even multidrug-resistant microorganisms, thereby increasing the risk of death.

[0004] wild chrysanthemum ( Chrysanthemum indicum *Chrysanthemum indicum* (L.), a member of the Asteraceae family, is a perennial plant that has been used as a traditional medicine in China for over 2000 years, widely used to treat pemphigus, swelling, pain, and lymph node tuberculosis. To date, more than 190 chemical components have been isolated and identified from this plant, including flavonoids, terpenes, phenylpropanoids, and phenolic acids. Essential oil (EO) is a concentrated hydrophobic liquid with a volatile aroma. It is formed from aromatic plants as secondary metabolites and consists of low-molecular-weight terpenes and terpenoid compounds, as well as other aromatic and aliphatic components. Combination drugs offer advantages such as multiple components, multiple targets, resistance to drug resistance, low adverse reactions, and relatively small effective doses, making it an important strategy for developing new drugs to treat diseases with complex etiologies. Currently, no research has shown whether the key active components in wild chrysanthemum essential oil have a therapeutic effect on acute lung injury; therefore, this invention is proposed. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to propose the use of the effective components of wild chrysanthemum essential oil in the treatment of acute lung injury.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] This invention proposes the application of the active ingredients of wild chrysanthemum essential oil in the preparation of drugs for the prevention and treatment of acute lung injury (ALI). The active ingredients of wild chrysanthemum essential oil include one or more of β-farnesene, 4-terpene alcohol, hesperidin, chrysanthemum alcohol, and carvacrol.

[0008] β-Farnese: CAS No. 18794-84-8.

[0009] 4-Terpene alcohol: CAS number 562-74-3.

[0010] Hesperene: CAS number 489-39-4.

[0011] Chrysanthemol: also known as 2,2-dimethyl-3-(2-methylpropenyl)cyclopropanol, CAS number 5617-92-5.

[0012] Carvacrol: CAS No.: 30460-92-5.

[0013] Preferably, the active ingredients of the wild chrysanthemum essential oil include 4-terpene alcohol, wild chrysanthemum alcohol, and carvone; the molar ratio of 4-terpene alcohol, wild chrysanthemum alcohol, and carvone is (0.05~0.4):(0.01~0.2):(0.4~1.0); more preferably (0.1~0.3):(0.015~0.1):(0.5~0.8); and even more preferably 0.295:0.05:0.655.

[0014] More preferably, the active ingredients of the wild chrysanthemum essential oil are composed of 4-terpene alcohol, wild chrysanthemum alcohol and carvone.

[0015] Preferably, the active ingredients of the wild chrysanthemum essential oil include 4-terpene alcohol, hesperidin, and carvacrol; the molar ratio of 4-terpene alcohol, hesperidin, and carvacrol is (0.2~1.0):(0.01~0.1):(0.1~0.5); more preferably (0.3~0.8):(0.04~0.08):(0.2~0.4); even more preferably 0.552:0.06:0.388.

[0016] More preferably, the active ingredients of the wild chrysanthemum essential oil are composed of 4-terpene alcohol, hesperidin and carvacrol.

[0017] Preferably, the active ingredients of the wild chrysanthemum essential oil include 4-terpene alcohol and carvone; the molar ratio of 4-terpene alcohol to carvone is (0.1~0.6):(0.3~1.0); more preferably (0.2~0.5):(0.4~0.8); and even more preferably 0.355:0.645.

[0018] More preferably, the active ingredients of the wild chrysanthemum essential oil consist of 4-terpene alcohol and carvacrol.

[0019] Preferably, the active ingredients of the wild chrysanthemum essential oil include β-farnesene, 4-terpene alcohol, hesperidin, and carvacrol; the molar ratio of β-farnesene, 4-terpene alcohol, hesperidin, and carvacrol is (0.05~0.5):(0.1~1.0):(0.01~0.1):(0.1~0.8); more preferably (0.1~0.3):(0.2~0.6):(0.03~0.08):(0.3~0.6); even more preferably 0.136:0.401:0.053:0.410.

[0020] More preferably, the active ingredients of the wild chrysanthemum essential oil are composed of β-farnesene, 4-terpene alcohol, hesperidin and carvacrol.

[0021] Preferably, the active ingredients of the wild chrysanthemum essential oil include hesperidin, chrysperidin alcohol, and carvacrol; the molar ratio of hesperidin, chrysperidin alcohol, and carvacrol is (0.05~0.5):(0.1~0.5):(0.1~1.0); more preferably (0.1~0.3):(0.2~0.3):(0.4~0.8); even more preferably 0.155:0.247:0.599.

[0022] More preferably, the active ingredients of the wild chrysanthemum essential oil are composed of hesperidin, chrysanthemum alcohol and carvacrol.

[0023] Preferably, the acute lung injury includes lipopolysaccharide-induced acute lung injury.

[0024] Preferably, the drug may contain medically acceptable excipients added to the active ingredient of wild chrysanthemum essential oil; or the active ingredient of wild chrysanthemum essential oil may be mixed with other drugs that have preventive or therapeutic effects on lipopolysaccharide-induced acute lung injury, with or without the addition of medically acceptable excipients.

[0025] Preferably, the drug is administered by injection or intraperitoneal injection.

[0026] The present invention also proposes the application of the above-mentioned active ingredients of wild chrysanthemum essential oil in the preparation of drugs for the prevention and treatment of acute respiratory distress syndrome (ARDS).

[0027] This invention also proposes the application of the above-mentioned active ingredients of wild chrysanthemum essential oil in the study of the mechanism of improving cell viability and reducing reactive oxygen species (ROS) levels in a lipopolysaccharide-induced airway epithelial cell injury model.

[0028] This invention also proposes the application of the above-mentioned active ingredients of wild chrysanthemum essential oil in the study of the mechanism of stabilizing mitochondrial membrane potential and regulating cytoskeleton structure in a lipopolysaccharide-induced airway epithelial cell injury model.

[0029] This invention also proposes the application of the above-mentioned active ingredients of wild chrysanthemum essential oil in the study of the mechanism of improving the pathological damage of lung tissue in a lipopolysaccharide-induced acute lung injury model in mice.

[0030] This invention also proposes the application of the above-mentioned active ingredients of wild chrysanthemum essential oil in the study of the mechanism of inhibiting the expression of inflammatory mediators in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide-induced mouse acute lung injury model.

[0031] The inflammatory mediators in the bronchoalveolar lavage fluid (BALF) include, but are not limited to, interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α).

[0032] The beneficial effects of this invention are as follows: (1) The active ingredients of wild chrysanthemum essential oil proposed in this invention can regulate cell viability and ROS levels: In an acute lung injury model, the active ingredients of wild chrysanthemum essential oil can significantly regulate the survival status of lung epithelial cells and improve the phenomenon of decreased cell viability. At the same time, it can effectively inhibit the excessive generation of reactive oxygen species (ROS), reduce the level of oxidative stress, thereby alleviating oxidative stress-mediated cell damage and playing an important protective role in maintaining lung tissue cell homeostasis.

[0033] (2) The active ingredients of wild chrysanthemum essential oil proposed in this invention can stabilize mitochondrial membrane potential and regulate cytoskeleton structure: Research results show that the active ingredients of wild chrysanthemum essential oil can effectively maintain the stability of mitochondrial membrane potential (ΔΨm) and alleviate mitochondrial dysfunction under acute lung injury conditions. At the same time, it can regulate cytoskeleton structure, reduce F-actin rearrangement disorder, and maintain the integrity of cell morphology and barrier function, thereby playing a protective role at the substructural level of cells.

[0034] (3) The active ingredients of wild chrysanthemum essential oil proposed in this invention can protect the structural integrity of lung tissue: In a mouse model of acute lung injury, the combination of active ingredients of wild chrysanthemum essential oil can significantly improve the pathological damage of lung tissue. By reducing pathological changes such as alveolar structural destruction, inflammatory cell infiltration and interstitial edema, it effectively maintains the structural integrity of lung tissue and plays a protective role against acute lung injury at the histological level.

[0035] (4) The active ingredients of wild chrysanthemum essential oil proposed in this invention can inhibit inflammatory mediators: In mice with acute lung injury, the combination of active ingredients of wild chrysanthemum essential oil can significantly inhibit the expression of inflammatory mediators in bronchoalveolar lavage fluid (BALF). It effectively reduces the level of inflammatory mediators in the body, alleviates the systemic inflammatory response caused by acute lung injury, and provides a positive and effective role in improving the pathological state of acute lung injury.

[0036] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0037] Figure 1 EdU staining was used to analyze the growth activity of BEAS-2B cells in different treatment groups in Example 1 of this invention (A); quantitative fluorescence analysis of the activity of BEAS-2B cells in different treatment groups (B). Figure 2 The first image shows the fluorescence image of ROS production in cells after (DCFH-DA) staining in Example 1 of this invention (A); the second image shows the fluorescence quantitative analysis of ROS levels in BEAS-2B cells from different treatment groups (B). Figure 3 The images show fluorescence images (A) of mitochondrial membrane potential in BEAS-2B cells stained with dye (JC-1) in Example 1 of this invention; and quantitative fluorescence images (B) of mitochondrial membrane potential in cells from different treatment groups. Figure 4 Analysis of the integrity of the cytoskeleton stained with Phalloidin in different treatment groups in Example 1 of this invention; Data are presented as mean ± standard deviation (n = 3); p<0.05, p<0.01, p<0.001, p<0.0001; Figure 5 A schematic diagram (A) of the dexamethasone (DEX) / effective drug component combination treatment in the LPS-induced ALI mouse model of the present invention; a representative image of H&E staining of lung tissue (B); Figure 6 The pathological assessment of lung tissue injury in Example 2 of this invention is scored (n = 6). Figure 7 The wet-to-dry weight ratio of mouse lung tissue in Example 2 of this invention (n = 6); Figure 8MPO activity in mouse lung tissue in Example 2 of this invention (n = 6) Figure 9 The protein concentration in the bronchoalveolar lavage fluid (BALF) of mice in Example 2 of this invention (n = 6); Figure 10 The expression levels of IL-6, IL-1β and TNF-α in BALF in Example 2 of the present invention (n = 6); Data are expressed as mean ± standard deviation (n = 6); ####P<0.0001 compared with the control group, p<0.05, p<0.01, p<0.001, p<0.0001 compared with the model group. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0039] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0040] Experimental cells: Human bronchial epithelial cells (BEAS-2B) were provided by the American Type Culture Collection (ATCC, USA).

[0041] Chemicals and reagents: Lipopolysaccharide (O55:B5) was purchased from Sigma-Aldrich; dexamethasone was purchased from YuanYe Biotechnology Co., Ltd.; the BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd.; β-farnesene, 4-terpene alcohol, hesperidin, chamomile alcohol, and carvacrol standards were all purchased from Shanghai Aladdin Biotechnology Co., Ltd., and dissolved in sterile DMSO solvent. Dexamethasone (DEX) was purchased from YuanYe Biotechnology Co., Ltd. (Shanghai, China). DMEM medium, 0.05% trypsin, PBS solution, fetal bovine serum (FBS), 1% penicillin, and streptomycin were purchased from Gibco (Grand Island, USA). Reactive oxygen species assay kits, EdU assay kits, DAPI, Actin-Tracker Red, and JC-1 kits were purchased from Beyotime Biotechnology Co., Ltd.; ELISA kits for IL-6, TNF-α, and IL-1β were purchased from Beyotime Biotechnology Co., Ltd. Deionized water was prepared using the Millipore Milli-Q water system. The ELISA kits for IL-6, TNF-α, and IL-1β were purchased from Beyotime Biotechnology Co., Ltd.

[0042] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0043] Example 1: Study on the protective effect of active ingredients in wild chrysanthemum essential oil on lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Wild chrysanthemum essential oil contains a variety of components. In the early screening studies, the applicant team found that wild chrysanthemum essential oil contains β-farnesene, 4-terpene alcohol, hesperidin, chrysanthemum alcohol, and carvone. These components may be candidate drugs or potential drugs for acute lung injury, and a series of studies were conducted on them.

[0044] Table 1: Candidate Drugs

[0045] The structural formulas of candidate drugs E1, E4, E8, E11, and E13 are shown below:

[0046] Logarithmic growth phase BEAS-2B cells were seeded into 96-well plates with black walls and clear bottoms (5000-8000 cells per well). The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C for 24 hours in 5% CO2. Then, the following methods were used: Treatment group LPS: LPS (1 μg / mL) Treatment group DEX: LPS (1 μg / mL) + dexamethasone (DEX) Treatment group E1: LPS (1 μg / mL) + candidate drug β-farnesene (E1) Treatment group E4: LPS (1 μg / mL) + candidate drug 4-terpene alcohol (E4) Treatment group E8: LPS (1 μg / mL) + candidate drug hesperidin (E8) Treatment group E11: LPS (1 μg / mL) + candidate drug chrysanthemum alcohol (E11) Treatment group E13: LPS (1 μg / mL) + candidate drug carvacrol (E13) Cells were treated for 24 hours. A blank control group (CON) was set up.

[0047] 1.1 EdU Detection: The growth activity of BEAS-2B cells after each treatment group was analyzed using EdU assay, and the specific procedure is as follows: Cells were incubated with EdU-containing medium. After removing the EdU-containing medium, the cells were washed with PBS buffer. Cells were fixed with formaldehyde to retain the labeled EdU. Next, the cells were permeabilized with an appropriate permeabilization buffer. Then, a click chemistry mixture containing a fluorescent probe was added to the fixed, permeabilized cells, and the cells were incubated for 30 minutes. Finally, the cells were washed with buffer to remove unreacted click chemistry reagents, and the cells were analyzed using a PerkinElmer Operetta CLS system for high-content imaging.

[0048] The results are as follows Figure 1 As shown, in the untreated control group, BEAS-2B cells exhibited a high EdU positivity rate, suggesting that they have good DNA synthesis capacity and proliferation activity.

[0049] In contrast, stimulation with lipopolysaccharide (LPS) significantly reduced the proportion of EdU-positive cells, indicating that LPS treatment substantially inhibited DNA synthesis and cell proliferation in BEAS-2B cells. This result suggests that inflammatory stimulation can directly lead to impaired airway epithelial cell proliferation, which is highly consistent with the pathological characteristics of massive release of inflammatory mediators and decreased epithelial repair capacity during acute lung injury. This further demonstrates that this in vitro model can effectively simulate the biological characteristics of epithelial cell dysfunction in ALI.

[0050] Based on this, after treating LPS-treated BEAS-2B cells with β-farnesene, 4-terpenol, hesperidin, chamomile alcohol, and carvacrol, the proportion of EdU-positive cells significantly increased compared to the LPS group, suggesting that the active ingredients of wild chrysanthemum essential oil can alleviate the LPS-induced cell proliferation inhibition effect to a certain extent and promote the recovery of cell DNA synthesis and growth capacity. These results indicate that β-farnesene, 4-terpenol, hesperidin, chamomile alcohol, and carvacrol may exert a protective effect on the proliferative function of damaged airway epithelial cells by improving the inflammatory microenvironment or regulating cell cycle-related signaling pathways.

[0051] 1.2 Detection of Reactive Oxygen Species (ROS): Intracellular reactive oxygen species (ROS) levels were measured using the DCFH-DA fluorescent probe. BEAS-2B cells from each treatment group were incubated at 37°C for 30 minutes in the dark with 10 μM DCFH-DA (dissolved in serum-free, phenol red-free DMEM medium). After incubation, cells were washed twice with PBS, replaced with fresh phenol red-free medium, and then subjected to high-content imaging. Cell nuclei were stained with Hoechst 33342 to assist in cell segmentation.

[0052] Oxidative stress is a key pathological mechanism driving the occurrence and progression of acute lung injury / acute respiratory distress syndrome (ALI / ARDS), often interacting with inflammatory responses and synergistically amplifying lung tissue damage. Under physiological conditions, adequate production of reactive oxygen species (ROS) is crucial for maintaining cell signal transduction, metabolic homeostasis, and normal physiological functions. However, under oxidative stress, excessive ROS production and accumulation, exceeding the clearance capacity of the intracellular antioxidant defense system, triggers a series of pathological changes. Excessive ROS can induce cell membrane lipid peroxidation, leading to the destruction of unsaturated fatty acid structures in the membrane, thereby reducing membrane fluidity and increasing membrane permeability; it can also activate various inflammation-related signaling pathways, upregulate the expression of pro-inflammatory cytokines and adhesion molecules, promote the recruitment of inflammatory cells, and ultimately exacerbate tissue damage and pulmonary edema. Therefore, inhibiting excessive ROS production and alleviating oxidative stress are considered important strategies for intervening in ALI / ARDS. This invention uses a reactive oxygen species detection kit to detect the intracellular ROS level in LPS-induced BEAS-2B cells after drug intervention.

[0053] The results are as follows Figure 2 As shown, compared with the control group, ROS in BEAS-2B cells was significantly accumulated after LPS stimulation, which was manifested as a strong green fluorescent signal, and the difference was statistically significant (P<0.05), suggesting that LPS can significantly induce oxidative stress in airway epithelial cells.

[0054] In contrast, treatment with β-farnesene, 4-terpene alcohol, hesperidin, chamomile alcohol, and carvone significantly reduced the intensity of green fluorescence in cells and decreased the ROS level, indicating that the active ingredients of wild chrysanthemum essential oil can effectively inhibit LPS-induced excessive ROS production.

[0055] The above results suggest that the active ingredients in wild chrysanthemum essential oil may significantly alleviate oxidative stress through their free radical scavenging or antioxidant activities, thereby reducing LPS-induced airway epithelial cell damage to some extent, providing experimental evidence for its potential application in the prevention and treatment of acute lung injury / acute respiratory distress syndrome (ALI / ARDS).

[0056] 1.3 Mitochondrial membrane potential detection: BEAS-2B cells treated in each group were incubated with JC-1 stain for 20 minutes, washed with culture medium, and then observed using a high-content imaging system. Under normal mitochondrial membrane potential, JC-1 forms red fluorescent aggregates; when the membrane potential decreases, JC-1 transforms into green fluorescent monomers, indicating mitochondrial damage.

[0057] (Mitochondrial dysfunction is considered one of the important pathological bases driving the progression of lung injury-related diseases. As the main site of intracellular reactive oxygen species production, mitochondria play a central role in regulating cellular energy metabolism, redox homeostasis, and cell fate determination. Normal mitochondrial membrane potential (ΔΨm) is crucial for maintaining the structural integrity and functional stability of mitochondria, and its stability is essential for maintaining normal cellular physiological activities. Under pathological conditions, when cells are damaged by inflammation or oxidative stress, ΔΨm often decreases in the early stages of apoptosis, thereby triggering mitochondrial pathway-mediated cell damage or apoptosis. To further explore the effects of drugs on mitochondrial function, this invention used the JC-1 fluorescent probe to detect LPS-induced changes in mitochondrial membrane potential. JC-1 exists in polymeric form under high membrane potential conditions, exhibiting red fluorescence; while under decreased membrane potential, it exists in monomeric form, exhibiting green fluorescence. Therefore, the red / green fluorescence intensity ratio can reflect the degree of mitochondrial membrane polarization.)

[0058] The results are as follows Figure 3 As shown in Figure A, compared with the control group, LPS treatment significantly reduced red fluorescence in cells while significantly enhanced green fluorescence, and the red / green fluorescence intensity ratio decreased significantly, suggesting that LPS can lead to mitochondrial membrane potential depolarization and mitochondrial function impairment.

[0059] like Figure 3As shown in Figure B, in contrast, after treatment with β-farnesene, 4-terpenol, hesperidin, chamomile alcohol, and carvone, the trend of LPS-induced decrease in red fluorescence and increase in green fluorescence was suppressed to varying degrees, and the red / green fluorescence intensity ratio rebounded significantly. This indicates that the active ingredients of wild chrysanthemum essential oil can effectively maintain the stability of mitochondrial membrane potential and alleviate LPS-induced mitochondrial dysfunction.

[0060] The above results suggest that it has a protective effect against LPS-induced lung injury, and its mechanism of reducing inflammatory response may be closely related to inhibiting mitochondrial functional damage and regulating mitochondrial-related pathways.

[0061] 1.4 Cytoskeleton Detection: BEAS-2B cells treated in each group were fixed with 4% paraformaldehyde for 15 minutes, then permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA. Staining was performed using Actin-Tracker (Beyotime) at room temperature in the dark for 30 minutes, followed by DAPI nuclear staining. Images were acquired using a high-content imaging system.

[0062] (Phallooidin staining was used to visualize the distribution of the cytoskeleton to reflect the integrity of the cytoskeleton under LPS-induced damage. Phalloidin specifically binds to actin filaments (F-actin), and the cytoskeleton structure is clearly displayed through fluorescence signals. When cells are stimulated by lipopolysaccharide (LPS), inflammatory signals activate downstream pathways through the TLR4 receptor, triggering an intracellular signaling cascade that ultimately leads to the rearrangement and depolymerization of F-actin. Multiple studies have shown that LPS can promote the depolymerization and recombination of the F-actin tandem structure, resulting in the disruption of the cytoskeleton architecture and significant changes in cell morphology. This process is closely related to the dysregulation of the barrier function of lung epithelial / endothelial cells.)

[0063] The results are as follows Figure 4As shown, LPS treatment led to significant depolymerization and disruption of F-actin, with the cytoskeleton exhibiting a broken and sparse distribution. Cell morphology changed from a regular, spreading pattern to a contracted or irregular one, characteristics consistent with cell damage patterns observed in acute lung injury (ALI) models. In the pathological progression of ALI, cytoskeleton remodeling-induced morphological changes and the loosening of cell-cell and cell-matrix connections further exacerbate epithelial / endothelial barrier dysfunction, increasing intercellular spaces and permeability, thereby promoting fluid exudation and pulmonary edema. More importantly, treatment with β-farnesene, 4-terpene alcohol, hesperidin, chamomile alcohol, and carvacrol, to some extent, maintained the integrity of F-actin and improved cell morphology, suggesting that they may exert a structural protective effect by stabilizing the cytoskeleton network, mitigating cell barrier imbalance caused by inflammatory damage.

[0064] Example 2: Treatment of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice with a combination of active ingredients (ECC) from wild chrysanthemum essential oil. 1. Laboratory animals Male C57BL / 6J mice, SPF grade, 6–8 weeks old, weighing (20±2) g, were purchased from Nanjing Jicui Pharmaceutical Co., Ltd. (Nanjing, China). Prior to the experiment, these animals were fed a standard diet and water for at least one week. They were also acclimatized to a 12-hour light / 12-hour dark cycle, with stable temperature (25±2℃) and humidity (50±5%). We took measures to minimize the number of animals used in the study and to reduce their suffering, including the use of anesthesia during surgery. We followed the three Rs (replacement, reduction, and refinement) principle in designing and implementing this invention.

[0065] 2. Lipopolysaccharide-induced acute lung injury model and treatment Fifty-four mice were randomly divided into nine groups (n=6 in each group): Control group (CON) Model Group (MOD) Positive drug control group: Dexamethasone group (DEX) Positive control group: Wild chrysanthemum essential oil group (EO); the wild chrysanthemum essential oil is a mixture of essential oils obtained by steam distillation.

[0066] Wild chrysanthemum active ingredient combination drug group: (ECC1, ECC2, ECC3, ECC4, ECC5).

[0067] The specific components and molar ratios of the active ingredient composition of wild chrysanthemum (ECC1, ECC2, ECC3, ECC4, ECC5) are shown in Table 2: Table 2

[0068] Except for the CON group, all mice were intraperitoneally injected with 10 mg / kg LPS (dissolved in physiological saline, volume 200 μL). The drug-treated groups received an intraperitoneal injection of 200 μL (10 mg / kg) of the drug solution 1 hour after modeling. Mice in the dexamethasone group received an intraperitoneal injection of 200 μL (10 mg / kg) of dexamethasone. Mice in the CON group received an intraperitoneal injection of an equal volume of physiological saline, and 24 hours after modeling, received an intraperitoneal injection of amobarbital solution (35 mg / kg). Serum, bronchoalveolar lavage fluid, and lung tissue were collected for further studies.

[0069] 3. Histopathological analysis 3.1 Histopathological sections Lung tissue was harvested after euthanasia and fixed with 10% neutral formaldehyde solution for 24 h, followed by dehydration with ethanol at gradient concentrations of 70%, 80%, 90%, 95%, and 100%. After clearing with xylene (Solarbio, China), the lung tissue was embedded in paraffin and cut into 5 μm sections. The sections were treated with xylene for 4 minutes, differentiated in hydrochloric acid-ethanol for 10 minutes, rinsed with eosin (Solarbio, China) for 2 minutes, fixed with neutral gel, and observed for lung injury and inflammatory cell infiltration using a slide scanner (3DHISTECH, Hungary).

[0070] (Histopathological sections are an important means of evaluating the degree of tissue damage and inflammatory response. To clarify the ameliorative effect of drugs on the pathological changes in lung tissue of LPS-induced ALI mice, this invention performed hematoxylin-eosin (HE) staining analysis on the lung tissue of mice in each group.) A schematic diagram of the DEX / effective drug combination treatment in the LPS-induced ALI mouse model is shown below. Figure 5 As shown in A; HE staining results show (e.g.) Figure 5As shown in Figure B), the lung tissue of mice in the Control group was intact, with regular alveolar morphology, clear alveolar cavity outlines, thinner alveolar septa, and no obvious edema or inflammatory cell infiltration in the lung interstitium, indicating that the lung tissue was in a normal physiological state. Compared with the Control group, the lung tissue of mice treated with LPS showed obvious pathological damage, mainly manifested as a large number of inflammatory cells such as neutrophils and macrophages infiltrating the lung interstitium and alveolar spaces, severe destruction of alveolar structure, obvious collapse of alveolar cavities, significant thickening of alveolar walls, and varying degrees of congestion and edema, indicating that LPS successfully induced typical ALI pathological changes. After intervention with ECC 10 mg / kg and the positive control dexamethasone (DEX, 10 mg / kg), the pathological damage of mouse lung tissue was improved to varying degrees. Specifically, the infiltration of inflammatory cells in the lung interstitium and alveolar spaces was significantly reduced, the alveolar structure tended to be intact, the alveolar septa were thinned, and the degree of edema and congestion in the lung tissue was reduced (e.g., Figure 5 (As shown in Figure B). Among them, the ECC1 drug group showed the most significant effect in improving the structural integrity of lung tissue and inhibiting inflammatory infiltration. Its pathological improvement was better than that of other drug combination groups, and its overall efficacy was better than that of the DEX treatment group.

[0071] 3.2 ALI Pathological Score The injury criteria included features such as atelectasis, alveolar and interstitial inflammation, hemorrhage, edema, necrosis, and overinflation. The ALI pathological scoring system was used to quantitatively analyze the degree of lung tissue injury in each group. The score was divided into four grades according to the degree of injury: Grade 0 (no injury), Grade 1 (≥25%), Grade 2 (≥50%), Grade 3 (≥75%), and Grade 4 (diffuse injury).

[0072] Lung injury score results as follows Figure 6 As shown, the lung tissue damage score of mice in the LPS group was significantly higher than that in the Control group (P<0.001), further validating that intraperitoneal injection of LPS can successfully establish an ALI mouse model. Compared with the LPS group, the lung tissue pathological scores of mice were significantly reduced after intervention with ECC 10 mg / kg and DEX 10 mg / kg (P<0.001), suggesting that the above intervention measures can effectively alleviate the degree of lung tissue damage in ALI mice.

[0073] 4. Wet-to-dry weight ratio of lung tissue Mice were weighed before euthanasia. Lung weight was also measured after lung removal. The formula for calculation was: Lung Index (mg / g) = Lung weight / Body weight. The left lung of the mouse was removed, rinsed with PBS, and its surface moisture was absorbed with filter paper. The lung tissue was then accurately weighed using an electronic analytical balance to obtain its wet weight. The lung tissue was dried in a dryer at 65°C for 48 hours, and then weighed to obtain its dry weight. The formula for calculation was: Lung wet-to-dry weight ratio (g / g) = Wet weight / Dry weight.

[0074] (The wet-to-dry weight ratio (W / D) is an important indicator for evaluating the degree of pulmonary edema.) The results are as follows Figure 7 As shown, compared with the Control group, the lung W / D ratio of ALI mice was significantly increased, indicating that there was significant pulmonary edema; while after intervention with the active ingredient combination of wild chrysanthemum essential oil (ECC), the lung W / D ratio of ALI mice was significantly decreased, indicating that the drug can effectively alleviate LPS-induced pulmonary edema.

[0075] 5. Detection of MPO activity in lung tissue Immediately after harvesting fresh mouse lung tissue, weigh it and place it in a pre-chilled centrifuge tube. Add 4000 μL of 50 mM HEPES buffer (0.65 g dissolved in 5 mL sterile water) and 50 μL of 10 mg / mL soybean trypsin inhibitor (STI) per gram of tissue. After adding a steel ball, homogenize the tissue for 10 minutes. Centrifuge the homogenate at 4 °C and 12,000 rpm for 10 minutes and discard the supernatant. Add 0.5% CTAC solution (4000 μL per gram of tissue) to the precipitate, homogenize again for 2 minutes, and centrifuge at 4 °C and 12,000 rpm for 5 minutes. Collect the supernatant as the MPO sample. After a 10-fold dilution, add 75 μL to each well of a 96-well plate. Then add the substrate working solution containing 3 mM TMB, 6 mM resorcinol, and 3% H2O2 to initiate the reaction. After the reaction is complete, add 2 M sulfuric acid to terminate the reaction, and measure the absorbance at 450 nm.

[0076] (The level of myeloperoxidase (MPO) activity in lung tissue can indicate the degree of neutrophil infiltration in the tissue. Abnormal activation and migration of neutrophils are one of the important characteristics of the development of ALI / ARDS. They can release a large number of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6. In order to investigate whether ECC can inhibit MPO activity in ALI, MPO activity in lung tissue was detected.)

[0077] The results are as follows Figure 8 As shown, compared with the control group, LPS-induced MPO activity in mouse lung tissue was significantly increased, while ECC intervention reduced LPS-induced MPO activity (P<0.001). This indicates that it can inhibit the recruitment and infiltration of neutrophils into the lungs.

[0078] 6. Bronchoalveolar lavage fluid (BALF) Mice were placed on the operating table in a supine position with their limbs immobilized to prevent movement. An incision was made in the skin over the trachea, exposing it using sterile surgical instruments. A small incision was made in the trachea using sterile scissors, and a sterile cannula was inserted. Physiological saline was injected into the lungs through the cannula, and the fluid was then aspirated back into a sterile syringe. The saline was gently injected and repeatedly aspirated to flush the lungs and collect the fluid. The fluid was collected in a sterile tube or container and centrifuged to remove cell debris. The supernatant was then stored... 80°C or process immediately for analysis.

[0079] Determination of total protein in BALF by BCA method Centrifuge the obtained BALF (600 g, 4℃, 10 min), and separate the supernatant for testing. Prepare standards to final concentrations of 0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 μg / μL according to the kit instructions. Each 400 μL standard concentration contains 0, 12.5, 25, 50, 100, 200, and 400 μL of the original standard, respectively. Mix BCA-A and BCA-B in a 50:1 volume ratio. Calculate the appropriate amount of BCA working solution based on the sample number and store at room temperature. Add each standard / sample and BCA working solution to a 96-well plate according to the instructions, mix thoroughly, place on a water bath rack, and maintain at 37℃ for 30 minutes. Set the microplate reader wavelength to 562 nm and obtain the absorbance (OD value) of the standards and BALF samples. A standard curve was plotted using the concentration and absorbance of the standard as variables. The formula was extracted, and the total protein concentration of BALF was obtained by substituting the absorbance of each sample as a variable into the formula.

[0080] (The total protein content in bronchoalveolar lavage fluid (BALF) can reflect changes in alveolar-capillary barrier permeability.) The results are as follows Figure 9 As shown, compared with the control group, the total protein content in the BALF of ALI mice was significantly increased, indicating increased vascular permeability and a large amount of plasma components leaking into the alveolar cavity; while after ECC intervention, protein exudation in BALF was significantly reduced, suggesting that the drug can improve the alveolar-capillary barrier function to a certain extent.

[0081] 7. Enzyme-linked immunosorbent assay (ELISA) to detect the expression levels of IL-6, IL-1β, and TNF-α in BALF. BALF solution was incubated at 4°C for 30 minutes and centrifuged at 3000 rpm. The concentrations of inflammatory factors in BALF were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Sanying) based on mouse interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). 100 μL of standard working solution or sample was added to the corresponding wells and incubated at 37°C for 90 minutes; 100 μL of biolabeled anti-tumor necrosis factor or interleukin-6 working solution was added and incubated at 37°C for 60 minutes; then 100 μL of enzyme-coupled working solution was added and incubated at 37°C for 30 minutes. 90 μL of substrate solution was added and incubated at 37°C for approximately 15 minutes. Finally, 50 μL of stop solution was added. Readings were immediately acquired at 450 nm using a microplate reader (Multiskan MK3, Thermo, USA).

[0082] Statistical analysis Graph Pad Prism software was used for statistical analysis of the groups involved. Data for each group are expressed as mean ± standard deviation. One-way ANOVA was used to compare data between multiple groups, and independent samples t-tests were used to compare data between two groups. P < 0.05 was considered statistically significant.

[0083] (The imbalance between pro-inflammatory cytokines and anti-inflammatory factors in the early inflammatory response of ALI is an important pathological feature, and the "cytokine storm" formed during the progression of the disease is one of the key factors leading to the high mortality rate of ALI.)

[0084] The results are as follows Figure 10As shown, compared with the control group, the levels of IL-6, IL-1β, and TNF-α in the BALF of ALI mice were significantly increased after LPS treatment (P<0.0001), with IL-6 at 142.7 ± 7.758 pg / mL (vs. 45.21 ± 3.461 pg / mL), IL-1β at 215.8 ± 9.874 pg / mL (vs. 73.81 ± 4.047 pg / mL), and TNF-α at 1100 ± 54.68 pg / mL (vs. 225.7 ± 16.42 pg / mL), indicating that the lung tissue of ALI mice was in a state of significant inflammatory imbalance. After ECC and DEX intervention, the levels of IL-6, IL-1β, and TNF-α in the BALF of ALI mice were significantly downregulated (P<0.01). Among them, the ECC1 group showed the most significant anti-inflammatory effect, with IL-6 at 91.43 ± 9.352 pg / mL, IL-1β at 124.8 ± 4.017 pg / mL, and TNF-α at 692.7 ± 43.63 pg / mL. Its effect in inhibiting the release of inflammatory factors was superior to other ECC examples and the DEX treatment group.

[0085] In summary, the above results indicate that the combined use of β-farnesene, 4-terpenoid alcohol, hesperidin, chamomile alcohol, and carvacrol can significantly improve the pathological damage of lung tissue in LPS-induced ALI mice, reduce pulmonary edema, inhibit neutrophil infiltration and excessive release of pro-inflammatory cytokines, thereby effectively alleviating the inflammatory response in the lungs. Its protective effect is closely related to the combination of β-farnesene, 4-terpenoid alcohol, hesperidin, chamomile alcohol, and carvacrol.

[0086] Example 3: Nitric Oxide (NO) Generation Experiment Mouse mononuclear macrophages RAW264.7 were provided by the Cell Bank of the Committee on Type Culture Collection, Chinese Academy of Sciences (CBTCCAS, Shanghai, China).

[0087] RAW264.7 cells were seeded at a density of 7.5 × 10³ cells per well in 96-well plates and cultured for 24 hours. Cells were then treated for 24 hours with LPS (1 μg / mL), LPS (1 μg / mL) combined with dexamethasone (DEX), and LPS combined with candidate drugs (β-farnesene, 4-terpenoid alcohol, hesperidin, chamomile alcohol, and carvacrol). After collecting the supernatant, 100 μL of the supernatant was mixed with 100 μL of Gliese reagent in a 96-well plate. The absorbance of the mixture was measured at 540 nm using a Multiskan MK3 microplate reader (Thermo Fisher Scientific, USA) and compared with sodium nitrate standard to quantitatively analyze the NO content in the supernatant. In the LPS-induced ALI in vitro model, NO reflects the level of inflammatory markers; detecting NO content was used to preliminarily screen the anti-inflammatory effects of drugs.

[0088] The NO inhibition rate is calculated using the following formula: Inhibition rate (%) = (NO content in LPS group) NO content in the treatment group / (NO content in the LPS group) NO content in the control group (×100%).

[0089] The combined effects of the drugs were evaluated using the Bliss independent model. The theoretical combined effect was calculated using the following formula: E ABC (expected) = E A + E B + E C E A E B E A E C E B E C + E A E B E C .

[0090] The formula for calculating the synergy index is: CI = E ABC (observed) / E ABC (expected).

[0091] Among them, E A E B E C E represents the inhibition rate when used individually. ABC(observed) represents the actual inhibition rate of the combined use. CI > 1 indicates synergistic effect, CI = 1 indicates additive effect, and CI < 1 indicates antagonistic effect.

[0092] Table 3

[0093] Table 4

[0094] The synergistic index (CI) calculated based on the inhibition rate is greater than 1, indicating a synergistic effect.

[0095] Based on the aforementioned active ingredients, a "composition" structure is constructed using a multilayer sensor mechanism. Proportion The "active" nonlinear mapping model, combined with an improved genetic algorithm for reverse design, and training with multiple random seeds to improve prediction reliability, ultimately selected several stable and efficient dosage combinations, including ECC1, ECC2, ECC3, ECC4, and ECC5. This demonstrates that specific dosage ratios are necessary to achieve the best results.

[0096] Example 4: The difference between this embodiment and Embodiment 2 is that the molar ratio of the components in the wild chrysanthemum active ingredient composition drug group is slightly adjusted.

[0097] Table 5

[0098] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the active ingredients of wild chrysanthemum essential oil in the preparation of drugs for the prevention and treatment of acute lung injury, characterized in that, The active ingredients of the wild chrysanthemum essential oil include one or more of β-farnesene, 4-terpene alcohol, hesperidin, chrysanthemum alcohol, and carvacrol.

2. The application according to claim 1, characterized in that, The active ingredients of the wild chrysanthemum essential oil include 4-terpenol, wild chrysanthemum alcohol and carvacrol; the molar ratio of 4-terpenol, wild chrysanthemum alcohol and carvacrol is (0.05~0.4):(0.01~0.2):(0.4~1.0).

3. The application according to claim 1, characterized in that, The active ingredients of the wild chrysanthemum essential oil include 4-terpene alcohol, hesperidin and carvacrol; the molar ratio of 4-terpene alcohol, hesperidin and carvacrol is (0.2~1.0):(0.01~0.1):(0.1~0.5).

4. The application according to claim 1, characterized in that, The active ingredients of the wild chrysanthemum essential oil include 4-terpene alcohol and carvacrol; the molar ratio of 4-terpene alcohol to carvacrol is (0.1~0.6):(0.3~1.0).

5. The application according to claim 1, characterized in that, The active ingredients of the wild chrysanthemum essential oil include β-farnesene, 4-terpenol, hesperidin and carvacrol; the molar ratio of β-farnesene, 4-terpenol, hesperidin and carvacrol is (0.05~0.5):(0.1~1.0):(0.01~0.1):(0.1~0.8).

6. The application according to claim 1, characterized in that, The active ingredients of the wild chrysanthemum essential oil include hesperidin, chrysperidin alcohol and carvacrol; the molar ratio of hesperidin, chrysperidin alcohol and carvacrol is (0.05~0.5):(0.1~0.5):(0.1~1.0).

7. The application according to claim 1, characterized in that, The drug may have medically acceptable excipients added to the active ingredient of wild chrysanthemum essential oil; or the active ingredient of wild chrysanthemum essential oil may be mixed with other drugs that have preventive or therapeutic effects on lipopolysaccharide-induced acute lung injury, with or without the addition of medically acceptable excipients.

8. The application according to claim 1, characterized in that, The drug is administered by injection or intraperitoneal injection.

9. The application of the active ingredients of wild chrysanthemum essential oil in the preparation of drugs for the prevention and treatment of acute respiratory distress syndrome, characterized in that, The active ingredients of the wild chrysanthemum essential oil include one or more of β-farnesene, 4-terpene alcohol, hesperidin, chrysanthemum alcohol, and carvacrol.

10. Applications of the active ingredients in wild chrysanthemum essential oil in any of the following: (1) Application in the study of the mechanism of improving cell viability and reducing reactive oxygen species level in lipopolysaccharide-induced airway epithelial cell injury model; (2) Application in the study of the mechanism of stabilizing mitochondrial membrane potential and regulating cytoskeleton structure in a lipopolysaccharide-induced airway epithelial cell injury model; (3) Application in the study of the pathological damage mechanism of lung tissue improvement in a lipopolysaccharide-induced acute lung injury model in mice; (4) Application in the study of the mechanism of inhibiting the expression of inflammatory mediators in bronchoalveolar lavage fluid in a lipopolysaccharide-induced mouse acute lung injury model.

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