Application of nauclea officinalis extract in preparation of medicine for treating chronic obstructive pulmonary disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a nauclea officinalis extract in preparation of a medicine for treating chronic obstructive pulmonary diseases. Experimental research shows that the nauclea officinalis extract is used for treating the chronic obstructive pulmonary disease, can inhibit lung inflammation of a patient suffering from the chronic obstructive pulmonary disease and improve the lung function and emphysema injury of the chronic obstructive pulmonary disease, can be used for preventing and treating the chronic obstructive pulmonary disease, and can be used for preventing and treating the chronic obstructive pulmonary disease. And a new strategy is provided for the development of medicines for treating chronic obstructive pulmonary diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of *Gnaphalium affine* extract in the preparation of drugs for treating chronic obstructive pulmonary disease. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory airway disease characterized by persistent, progressive airflow limitation. Its pathological basis is closely related to the enhanced abnormal inflammatory response of the airways and lung tissue to harmful particles or gases (such as tobacco smoke, air pollutants, and occupational dust). COPD is mainly caused by long-term chronic irritation, with core contributing factors including long-term smoking, air pollution, occupational dust (such as coal dust and chemicals), recurrent airway infections, and genetic factors (such as α1-antitrypsin deficiency). The core of COPD pathogenesis is chronic airway inflammation, characterized by infiltration of neutrophils, macrophages, and T lymphocytes, releasing inflammatory mediators such as interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α), triggering airway remodeling (goblet cell hyperplasia leading to mucus hypersecretion, smooth muscle hypertrophy, and fibrosis). An imbalance between proteases and antiproteases results in increased activity of proteolytic enzymes such as neutrophil elastase, while inhibitors such as α1-antitrypsin are insufficient, causing degradation of alveolar wall elastic fibers and emphysema, ultimately leading to irreversible airway stenosis and airflow limitation. Current clinical treatment mainly relies on bronchodilators and glucocorticoids, but long-term use of hormones may cause side effects such as increased risk of infection and osteoporosis, and the efficacy is limited for some patients. Therefore, there is an urgent need to develop a substance with both anti-inflammatory activity and high safety for the treatment of COPD. Summary of the Invention

[0003] Based on this, the present invention provides the application of *Echinopsis lanceolata* extract in the preparation of a drug for treating chronic obstructive pulmonary disease (COPD), in order to solve the current problem of the lack of related drugs for treating COPD.

[0004] The present invention adopts the following technical solution:

[0005] This invention provides the application of *Echinopsis lanceolata* extract in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0006] *Nauclea officinalis* Pierre ex Pitard., belonging to the Rubiaceae family and the *Nauclea* genus, has branches and bark with properties that clear heat and detoxify, reduce swelling and relieve pain. Clinically, it is used to treat inflammatory diseases such as colds, fever, bronchitis, and pneumonia. Its core active components include alkaloids (such as isoclavillarin lactam), flavonoids, and terpenes, which endow *Nauclea officinalis* extract with significant anti-inflammatory, antioxidant, antibacterial, and antiviral properties. To date, there are no research reports on the application of *Nauclea officinalis* and its active ingredients in the prevention and treatment of chronic obstructive pulmonary disease (COPD).

[0007] Furthermore, the extract of *Gnaphalium affine* is obtained by extraction using ethanol with a volume concentration of 70%-95% as a solvent.

[0008] Furthermore, the extraction method of the *Gnaphalium affine* extract is as follows: after drying and pulverizing the *Gnaphalium affine* trunk, add 5-10 times the mass of ethanol aqueous solution for reflux extraction. The reflux extraction time is 1-3 hours, the extraction is performed 1-3 times, and the extraction temperature is 70℃-85℃.

[0009] Furthermore, the main components of the *Cinnamomum camphora* extract include isovinctin lactam and / or the monoterpene indole alkaloid naucleficine B.

[0010] Furthermore, the separation and purification method of naucleficine B is as follows: The extract of *Gnaphalium affine* is extracted with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate extract is then added to a silica gel column for chromatography (cyclohexane-acetone, 9:1 → 5:5), collecting a total of 15 fractions. The insoluble powder from the fifth fraction is dissolved in DMSO and purified by preparative liquid chromatography (column: Cosmosil 5C). 18 -MS-II, 10×250mm, 5μm; mobile phase: acetonitrile-water (60:40); flow rate: 3mL / min), thus obtaining the naucleficine B.

[0011] Furthermore, the molecular formula of naucleficine B is C 22 H 20 N₂O₃ (mass-to-charge ratio m / z 361.1546, molecular ion type [M+H]) + Its chemical structure is shown in structural formula I:

[0012]

[0013] Furthermore, the naucleficine B is an orange amorphous powder with a single crystal size of 0.12 × 0.1 × 0.08 mm. 3 It belongs to the orthorhombic crystal system, with space group P212121; cell parameters:

[0014] Furthermore, the molecular formula of the isocinolone lactone is C 26 H 30 N₂O₈, its chemical structure is shown in structural formula II:

[0015]

[0016] Furthermore, the drug is used to suppress lung inflammation in patients with chronic obstructive pulmonary disease.

[0017] Furthermore, the drug is used to improve lung function in patients with chronic obstructive pulmonary disease.

[0018] Furthermore, the drug is used to improve emphysematous lesions in patients with chronic obstructive pulmonary disease.

[0019] Furthermore, the isoclavulanic acid lactam and / or naucleficine B can inhibit the activity of inducible nitric oxide synthase (iNOS) induced by CSE in RAW264.7 and THP-1 cells, and significantly reduce NO production induced by cigarette smoke extract (CSE).

[0020] Furthermore, the isoclavulanic acid lactam and / or naucleficine B can inhibit the transcription of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 genes in CSE-induced RAW264.7 and THP-1 cells, and significantly reduce the secretion of pro-inflammatory factors.

[0021] Furthermore, the isoclavulanic acid lactam and / or naucleficine B can inhibit the expression of MMP-9, MMP-12 and COX-2 proteins induced by CSE in RAW264.7 and THP-1 cells.

[0022] Furthermore, the isoclavulanic acid lactam and / or naucleficine B can significantly downregulate the phosphorylation levels of p65, IκBα, c-Jun, c-Fos, STAT1 and STAT3 in CSE-induced RAW 264.7 and THP-1 cells, and inhibit the activation of NF-κB, AP-1 and STAT transcription factors.

[0023] Furthermore, the isoclavulanic acid lactam and / or naucleficine B can inhibit the expression of p-JNK, p-p38, p-ERK1 / 2, TLR4 and MyD88 signaling molecules induced by CSE in RAW264.7 and THP-1 cells, inhibit the MAPK and TLR4 signaling pathways, and exert anti-inflammatory effects.

[0024] The present invention also discloses a pharmaceutical composition for treating chronic obstructive pulmonary disease, wherein the sole active ingredient of the pharmaceutical composition is an extract of *Echinochloa crus-galli*.

[0025] Furthermore, the content of the extract of *Gnaphalium affine* in the pharmaceutical composition is 1 wt% to 99 wt%.

[0026] Furthermore, the dosage form of the pharmaceutical composition includes granules, tablets, capsules, oral liquids, mixtures, pills, ointments, creams, or transdermal absorption preparations.

[0027] Furthermore, the drug also includes other pharmaceutically acceptable excipients; these other pharmaceutically acceptable excipients include: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0028] The present invention has the following beneficial effects:

[0029] This invention is the first to discover that *Gnaphalium affine* extract can inhibit pulmonary inflammation in patients with chronic obstructive pulmonary disease (COPD), improve pulmonary function and emphysematous lesions in COPD, and can be used for the prevention and treatment of COPD. Furthermore, this invention is the first to isolate and identify a novel monoterpenoid indole alkaloid, naucleficine B, from *Gnaphalium affine* extract, with the molecular formula C. 22 H 20 N₂O₃ (mass-to-charge ratio m / z 361.1546, molecular ion type [M+H]) + In vitro therapeutic efficacy studies have shown that naucleficine B has good biocompatibility, significantly inhibiting CSE-induced inducible nitric oxide synthase (iNOS) activity in RAW 264.7 and THP-1 cells, significantly reducing CSE-induced NO production, decreasing pro-inflammatory cytokine secretion, inhibiting MMP-9, MMP-12, and COX-2 protein expression, significantly downregulating CSE-induced phosphorylation levels of p65, IκBα, c-Jun, c-Fos, STAT1, and STAT3 in RAW 264.7 and THP-1 cells, inhibiting the activation of NF-κB, AP-1, and STAT transcription factors, and simultaneously inhibiting CSE-induced expression of p-JNK, p-p38, p-ERK1 / 2, TLR4, and MyD88 signaling molecules in RAW 264.7 and THP-1 cells, inhibiting the MAPK and TLR4 signaling pathways, and exerting anti-inflammatory effects. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is the mass spectrum of naucleficine B.

[0032] Figure 2 The X-ray crystal structure of naucleficine B.

[0033] Figure 3 For naucleficine B 1 H-NMR spectrum.

[0034] Figure 4 For naucleficine B 13 C-NMR spectrum.

[0035] Figure 5 The graph shows the results of cytotoxicity testing (n=4); among them, Figure 5 In the figure, A represents the cell viability results of RAW 264.7 and THP-1 cells after 24 hours of treatment with naucleficine B; Figure 5 Figure B in the figure shows the cell viability results of RAW 264.7 and THP-1 cells after treatment with different concentrations of isovendilamide for 24 hours.

[0036] Figure 6 The figure shows the effects of isovinclinolactam and naucleficine B on NO production in cells (n=3). Figure 6 Figure A shows the effect of naucrite B on NO in CSE-induced RAW 264.7 and THP-1 cells. Figure 6 In the figure, B represents the NO inhibition rate curve of naucleficine B on CSE-induced RAW 264.7 cells and THP-1 cells; Figure 6 In the figure, C represents the effect of isovinctin lactam on NO in CSE-induced RAW 264.7 and THP-1 cells; Figure 6 In the figure, D represents the NO inhibition rate curve of isocinolone lactone on CSE-induced RAW 264.7 and THP-1 cells; Figure 6 The figure shows the effect of Dex on NO in CSE-induced RAW 264.7 and THP-1 cells. Figure 6F in the figure represents the NO inhibition rate curve of Dex on CSE-induced RAW 264.7 cells and THP-1 cells;

[0037] Figure 7 The figure shows the effect of isovinclinolactam and naucleficine B on the expression level of iNOS mRNA in cells (n=3); among them, Figure 7 In the figure, A represents the effect of naucrite B on iNOS mRNA expression induced by CSE in RAW 264.7 cells; Figure 7 In this context, B represents the effect of naucleficine B on CSE-induced iNOS mRNA expression in THP-1 cells. Figure 7 In this context, C represents the effect of isovincinolactam on iNOS mRNA expression in CSE-induced RAW 264.7 cells; Figure 7 In the figure, D represents the effect of isovincalactin-lactam on iNOS mRNA expression in CSE-induced THP-1 cells.

[0038] Figure 8 The effect of isovinctin lactam and naucleficine B on the expression of inflammatory factors in cells (n=3); Figure 8 Figure A shows the effect of naucleficine B on the expression levels of inflammatory factors in CSE-induced RAW 264.7 cells; Figure 8 The figure shows the effect of naucleficine B on the expression levels of inflammatory factors in CSE-induced THP-1 cells. Figure 8 In the figure, C represents the effect of isovinctin lactam on the expression levels of inflammatory factors in CSE-induced RAW 264.7 cells; Figure 8 In the figure, D represents the effect of isovincalactin-lactam on the expression levels of inflammatory factors in CSE-induced THP-1 cells.

[0039] Figure 9 The figure shows the effect of naucrite B on the expression of p-p65, p65, p-IκBα, and IκBα proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 9 In the diagram, A represents the Western blot image; Figure 9 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 9 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0040] Figure 10The figure shows the effect of isovincalactin-lactam on the expression of p-p65, p65, p-IκBα, and IκBα proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 10 In the diagram, A represents the Western blot image; Figure 10 B in the image represents the Western blot quantitative image in RAW264.7 cells; Figure 10 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0041] Figure 11 The figure shows the effect of naucleficine B on the expression of pc-Jun, c-Jun, pc-Fos, and c-Fos proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 11 In the diagram, A represents the Western blot image; Figure 11 B in the image represents the Western blot quantitative image in RAW264.7 cells; Figure 11 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0042] Figure 12 Figure 1 shows the effect of isovinctin lactone on the expression of pc-Jun, c-Jun, pc-Fos, and c-Fos proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 12 In the diagram, A represents the Western blot image; Figure 12 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 12 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0043] Figure 13 The effect of naucleficine B on the expression of p-stat1, stat1, p-stat3, and stat3 proteins in CSE-induced RAW 264.7 and THP-1 cells is shown in the figure (n=4); among them, Figure 13 In the diagram, A represents the Western blot image; Figure 13 B in the image represents the Western blot quantitative image in RAW264.7 cells; Figure 13 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0044] Figure 14 The figure shows the effect of isovinctin lactone on the expression of p-stat1, stat1, p-stat3, and stat3 proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 14 In the diagram, A represents the Western blot image; Figure 14 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 14C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0045] Figure 15 The effect of naucrite B on the expression of p-JNK, JNK, p-p38, p38, p-ERK1 / 2, and ERK1 / 2 proteins induced by CSE in RAW 264.7 and THP-1 cells is shown in the figure (n=4). Figure 15 In the diagram, A represents the Western blot image; Figure 15 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 15 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0046] Figure 16 Figure 1 shows the effect of isovinctin lactone on the expression of p-JNK, JNK, p-p38, p38, p-ERK1 / 2, and ERK1 / 2 proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4). Figure 16 In the diagram, A represents the Western blot image; Figure 16 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 16 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0047] Figure 17 The figure shows the effect of naucleficine B on the expression of MyD88 and TLR4 proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 17 In the diagram, A represents the Western blot image; Figure 17 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 17 C in the figure represents the quantitative protein blot diagram in THP-1 cells.

[0048] Figure 18 The figure shows the effect of isovincalactin-lactam on the expression of MyD88 and TLR4 proteins in CSE-induced RAW 264.7 and THP-1 cells (n=4); among them, Figure 18 In the diagram, A represents the Western blot image; Figure 18 B in the image represents the quantitative protein blot diagram in RAW 264.7 cells; Figure 18 C in the figure represents the quantitative protein blot diagram in THP-1 cells. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and 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 protection scope of the present invention.

[0050] Example 1: Efficacy verification of *Gnaphalium affine* extract in chronic obstructive pulmonary disease

[0051] 1. Preparation of Gallnut Extract

[0052] The trunk of the *Gnaphalium affine* tree was naturally dried and pulverized through a 60-mesh sieve. It was then refluxed with 7 times its weight of 95% ethanol aqueous solution for 2 hours, twice, at a temperature of 85°C. The extracts were combined to obtain the *Gnaphalium affine* extract, which was then concentrated under reduced pressure to a paste with a relative density of 1.10 for later use.

[0053] 2. Modeling and drug administration methods

[0054] (1) Experimental animals: 90 male Wistar rats, 6-8 weeks old, weighing 180-220g.

[0055] (2) Animal model establishment: COPD modeling method in smoking rats: Cigarettes (20 cigarettes / time) were placed in a smoke generator, and rats were placed in a nebulizer inhalation box with dimensions of 60 cm × 60 cm × 80 cm. After lighting the cigarettes, the smoke was injected into the inhalation box by automatic suction using a syringe, and the smoke had to be completely burned within five minutes. The inhalation was performed twice a day, morning and evening, for 30 minutes each time, with an interval of more than 4 hours, for 180 consecutive days.

[0056] (3) Grouping and Drug Administration: Rats were randomly divided into 6 groups (n=15), including a blank control group (healthy rats), a model control group (COPD model), high-, medium-, and low-dose groups of *Euphorbia pekinensis* extract, and an aminophylline tablet group. The high-, medium-, and low-dose groups of *Euphorbia pekinensis* extract were administered the prepared extract. Specifically, the high-dose group of *Euphorbia pekinensis* extract was administered at a dose of 0.444 g / kg / day, the medium-dose group at 0.222 g / kg / day, the low-dose group at 0.111 g / kg / day, and the aminophylline tablet group at 0.01 g / kg / day. Intervention began 90 days after modeling, with oral administration once daily for 90 days, 1 hour before smoke exposure. The blank control group and the model control group were given physiological saline.

[0057] 3. Testing Methods

[0058] Lung function tests: One hour after the last administration, forced vital capacity (FVC) and forced expiratory volume in 0.3 seconds (FEV1) were measured after quiet breathing. 0.3 ), FEV 0.3 / FVC parameter determination.

[0059] Emphysema index testing: Right upper and middle lung tissue was harvested, and 10% formalin solution was instilled into the right main bronchus until the right lower lung expanded. After the pleura flattened, the tissue was fixed in formalin for 24 hours, followed by HE staining for routine pathological examination. Emphysema was observed under a microscope. The average alveolar lining septum was measured; the average number of alveoli was counted; the area of ​​each field of view under the pathological image analysis system was measured, and the number of alveoli in that field of view was counted.

[0060] MAN = Number of alveoli in the field of view / Area of ​​the field of view; its value reflects the average alveolar density.

[0061] Bronchoalveolar lavage fluid (BALF) inflammatory factor and protease assay: The BALF was lavaged three times using 4 mL, 4 mL, and 2 mL of sterile saline via an indwelling intravenous catheter. A recovery rate greater than 80% was considered a qualified sample. After centrifugation at 1500 rpm for 10 min, the supernatant was collected, aliquoted, and frozen at -80°C. Cells were washed with pre-chilled Hanks' solution and classified for counting. The remaining fluid was used to prepare cell smears, stained with Wright-Giemsa, and then classified for counting. The supernatant was used to detect pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, TNF-α, MMP-9, and MMP-12 using ELISA.

[0062] 4. Experimental Results

[0063] (1) Lung function test results: The lung function test results are shown in Table 1. Compared with the blank control group, the FVC and FEV of rats in the model control group were significantly different. 0.3 FEV 0.3 The FVC and FEV1 were significantly reduced, indicating successful model establishment. Compared with the model group, the low, medium, and high dose groups of *Gnaphalium affine* extract showed significantly lower levels of lung function parameters FVC and FEV1. 0.3 FEV 0.3 Both FVC and FLVC were elevated. The results indicate that *Gnaphalium affine* extract can improve lung function and increase pulmonary ventilation in COPD model rats.

[0064] Table 1 Comparison of pulmonary function test results among groups

[0065] Group FVC / mL <![CDATA[FEV 0.3 / mL]]> <![CDATA[FEV 0.3 / FVC / %]]> Blank control group 7.56±0.27 6.29±0.19 83.20±0.72 Model control group <![CDATA[4.47±0.11 ## ]]> <![CDATA[2.14±0.14 ## ]]> <![CDATA[47.87±3.14 ## ]]> Low-dose group of gentian extract 4.6±0.15 <![CDATA[2.66±0.12 ** ]]> <![CDATA[57.82±0.76 ** ]]> Medium dose group of gentian extract <![CDATA[5.05±0.18 ** ]]> <![CDATA[3.06±0.10 ** ]]> <![CDATA[60.59±0.55 ** ]]> High-dose group of gentian extract <![CDATA[5.67±0.20 * ]]> <![CDATA[3.62±0.09 ** ]]> <![CDATA[63.84±0.57 ** ]]> Aminophylline tablets <![CDATA[5.13±0.24 ** ]]> <![CDATA[3.61±0.39 ** ]]> <![CDATA[70.37±4.45 ** ]]>

[0066] Note: Compared with the blank group: ## P<0.01; Compared with the model group: * P<0.05, ** P<0.01.

[0067] (2) Results of emphysema index detection: The results of emphysema index detection are shown in Table 2. In the model control group, the MLI of rats was significantly increased and the MAN was significantly decreased, indicating severe damage to the alveolar structure, indicating successful modeling. Compared with the model group, the MLI of the low, medium and high dose groups of *Eucommia ulmoides* extract was significantly decreased, while the MAN was significantly increased. Moreover, the average number of alveoli in the medium and high dose groups of *Eucommia ulmoides* extract was higher than that in the aminophylline tablet group. The results show that *Eucommia ulmoides* extract can effectively reverse alveolar structural damage and repair emphysematous lesions in COPD model rats.

[0068] Table 2 Comparison of MLI and MAN in lung tissue of rats in each group

[0069]

[0070]

[0071] Note: Compared with the blank group: # P<0.05; Compared with the model group: * P<0.05.

[0072] (3) Results of BALF inflammatory factors and proteases: The results of BALF inflammatory factor testing are shown in Table 3. Compared with the blank control group, the levels of pro-inflammatory factors IL-1β, IL-6, IL-8, TNF-α and proteases MMP-9 and MMP-12 in the BALF of rats in the model control group were significantly increased, confirming the surge in pulmonary inflammatory response after modeling and the successful modeling. Compared with the model group, the levels of pro-inflammatory factors IL-1β, IL-6, IL-8, TNF-α and proteases MMP-9 and MMP-12 in the low, medium and high dose groups of *Eucommia ulmoides* extract were significantly decreased, and the inhibitory effect of the high dose group of *Eucommia ulmoides* extract was significantly better than that of the aminophylline group. The results indicate that *Eucommia ulmoides* extract can inhibit the pulmonary inflammatory response in COPD model rats and target and reduce the expression levels of pro-inflammatory factors and proteases.

[0073] Table 3 Comparison of inflammatory factor levels in BALF of rats in different groups pg / mL)

[0074]

[0075] Note: Compared with the blank group: # P<0.05; Compared with the model group: * P<0.05.

[0076] The results of inflammatory cell expression are shown in Table 4. Compared with the blank control group, the total number of inflammatory cells, the proportion of neutrophils and macrophages in the BALF of the model control group increased significantly, while the proportion of lymphocytes decreased significantly, indicating successful modeling. Compared with the model group, the total number of inflammatory cells, the proportion of neutrophils and macrophages in the low, medium and high dose groups of *Eucommia ulmoides* extract decreased significantly, while the proportion of lymphocytes increased significantly. This indicates that *Eucommia ulmoides* extract can significantly reduce the total number of inflammatory cells, inhibit neutrophil infiltration, restore lymphocyte-dominated adaptive immune homeostasis, regulate the proportion of macrophages, and achieve therapeutic effects on COPD.

[0077] Table 4. Effects of *Eucommia ulmoides* extract on inflammatory cell expression in BALF of COPD rats.

[0078]

[0079]

[0080] Note: Compared with the blank group: # P<0.05, ## P<0.01; Compared with the model group: * P<0.05, ** P<0.01.

[0081] Example 2: Extraction, purification and identification of active ingredients from *Gnaphalium affine* extract

[0082] 1. Purification method

[0083] The extract obtained in Example 1 was suspended in water to prepare a suspension (the mass-to-volume ratio of extract to water was 1:10.22). At room temperature, it was extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, 20 times with each organic solvent, each time at a 1:1 volume ratio of organic solvent to suspension. The mixture was stirred with a glass rod, and after standing for 30 minutes to separate the layers, the organic layer was concentrated under reduced pressure at 45°C and a vacuum degree <0 MPa to obtain the petroleum ether, ethyl acetate, and n-butanol fractions. The extracted ethyl acetate fraction was initially separated by silica gel column chromatography, with a cyclohexane-acetone gradient elution (9:1→5:5). A total of 15 fractions were collected and sorted in ascending order of polarity.

[0084] (1) Purification of naucelficine B: The insoluble powder from the fifth fraction collected above was dissolved in DMSO and purified by preparative liquid chromatography. The chromatographic conditions were as follows: column: Cosmosil 5C 18-MS-II (10×250mm, 5μm); mobile phase: acetonitrile-water (60:40), flow rate: 3mL / min, to obtain compound naucleficine B (retention time 19.5min).

[0085] (2) Purification of isovincin lactam: The 14th fraction collected above was added to a silica gel column for separation. The mobile phase was chloroform-methanol gradient elution (100:0→0:100), and a total of 28 sub-fractions were obtained (named in order of separation: Fr.1~Fr.28). Fr.23 was added to a Sephadex LH-20 gel column and eluted with methanol to obtain 4 sub-fractions (named in order of separation: ad). Fraction b was added to preparative liquid chromatography for separation and purification. The chromatographic conditions were: column: Cosmosil 5C 18 MS-II (10×250mm, 5μm), mobile phase acetonitrile-water (30:70), flow rate 3mL / min, yielded the compound isovinctin lactam (retention time 17.6min).

[0086] 2. Naucelficine B Identification Method

[0087] (1) Mass spectrometry analysis: A Thermo LTQ Orbitrap XL mass spectrometer was used for analysis. The sample was dissolved in methanol and detected in positive ion mode. Mass calibration was performed using the internal standard method. The results are as follows: Figure 1 As shown, the molecular formula of naucleficine B was determined to be C by HRESIMS. 22 H 20 N₂O₃, [M+H] detected + Ion peak m / z 361.1546 (calculated value C) 22 H 21 N2O3 is 361.1547.

[0088] (2) X-ray single-crystal diffraction: A Rigaku Oxford Diffraction Supernova diffractometer (Cu Kα radiation) was used. Data was collected at a low temperature of 100.15 K. The crystal size was 0.12 × 0.1 × 0.08 mm. 3 The scanning range is 6.454°≤2θ≤136.464°. The results are as follows: Figure 2 As shown, naucleficine B(C 22 H 21 The single crystal size of N₂O₃ (molecular weight 360.40 g / mol) is 0.12 × 0.1 × 0.08 mm. 3It belongs to the orthorhombic crystal system, with space group P212121; cell parameters: A total of 14,593 reflection points were measured in the experiment, and after deduplication, 3,073 independent reflections were obtained (merging factor Rint = 0.0698, Rsigma = 0.0512); the final deviation factor R1 = 0.0540 (for diffraction points with I > 2σ(I)), the absolute configuration Flack parameter = 0.25(19), and the crystal structure data have been stored in the Cambridge Crystallography Data Center (CCDC 2395410).

[0089] (3) Nuclear magnetic resonance: NMR spectra were detected on a Bruker AV-600 spectrometer (Germany), with DMSO-d6 as the solvent. 1 H NMR at 600MHz and 13 The C NMR is 150MHz;

[0090] like Figure 3 As shown in Table 5, naucleficine B 1 H-NMR spectrum shows four aromatic protons [δ] H 7.60 (1H,d,J=7.9Hz,H-9), 7.46 (1H,d,J=7.9Hz,H-12), 7.23 (1H,t,J=7.9Hz,H-11) and 7.08 (1H,t,J=7.9Hz,H-10)] and two methylene protons [δ H The values ​​of 4.40 (2H,t,J = 6.6Hz,H-5) and 3.09 (2H,t,J = 6.6Hz,H-6) indicate the presence of a tetrahydro-β-carbamoline ring in its structure. Furthermore, 1 The H-NMR spectrum also showed an amino proton δ H 11.77 (1H, s, NH₁), four olefin protons δ H 8.27 (1H, d, J = 7.7 Hz, H-19), 7.83 (1H, d, J = 7.7 Hz, H-17), 7.46 (1H, t, J = 7.7 Hz, H-18), and 7.29 (1H, s, H-14), one methine proton δ H 5.89 (1H, s, H-22) and two methoxy protons δ H 3.31(6H,s,22-OCH3×2). Furthermore, such as... Figure 4 As shown in Table 5, naucleficine B 13 The C-NMR spectrum showed 22 carbon signals, including two methoxy groups, two methylene groups, nine methine groups, one carbonyl group, and eight other types of quaternary carbon signals, suggesting that naucleficine B is a monoterpenoid indole alkaloid.

[0091] Table 5. Isoclavulanic acid lactam and naucleficine B 1 H-NMR and 13 C-NMR data.

[0092]

[0093]

[0094] Example 3: Study on the in vitro therapeutic effect

[0095] 1. Experimental Methods

[0096] (1) Construction of cell inflammation model

[0097] Cell Culture and Treatment: RAW264.7 cells were cultured in DMEM medium, and THP-1 cells were cultured in RPMI 1640 medium, both supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 12-well plates and cultured at 37°C and 5% CO2 for 24 hours.

[0098] Preparation of Cigarette Smoke Extract (CSE): Daqianmen brand cigarettes (each containing 11.0 mg tar, 0.8 mg nicotine, and 13.0 mg carbon monoxide) were lit, and the cigarette smoke was collected and dissolved in 70% ethanol. After drying with nitrogen, the smoke was dissolved in DMSO to prepare a 100 mg / mL solution for later use.

[0099] Cell model construction: RAW 264.7 cells were pretreated with the test sample for 2 hours, and then incubated with 85 μg / mL CSE for 24 hours. Control group cells were only added with an equal volume of solvent (DMSO) and CSE.

[0100] THP-1 cells were treated with 160 nM PMA for 24 hours to induce differentiation into a macrophage-like phenotype. After pretreatment with the test sample for 1 hour, they were incubated with 10 μg / mL CSE for 12 hours. Control cells received only an equal volume of solvent and CSE. Dexamethasone (Dex) was used as a positive control at a concentration of 1.0 μM in RAW 264.7 cells and 0.5 μM in THP-1 cells.

[0101] (2) Detection method

[0102] 1) Cytotoxicity assay: To assess the cytotoxicity of the test samples, RAW 264.7 and THP-1 cells were seeded in 96-well plates (1×10⁻⁶ cells / well). 4Cells / well) were incubated overnight at 37°C. After treating with different concentrations of the test sample for 24 hours, the medium was replaced with 100 μL of serum-free medium and 10 μL of CCK-8 solution (purchased from Jingxin Biotechnology) was added. The mixture was incubated for 1 hour, and the absorbance (OD value) was measured at 450 nm. All data are expressed as mean ± standard error. * P<0.05, ** P<0.01, *** P<0.001.

[0103] 2) NO generation inhibition assay: NO generation inhibition: RAW 264.7 and THP-1 cells were treated with the test sample and stimulated with CSE according to the cell model construction method described above. Cells were lysed and centrifuged (12000 rpm, 4℃, 10 min) to obtain the supernatant. The supernatant was reacted with Griess reagent (purchased from Beyotime), and the absorbance was measured at 540 nm. NO2 was quantified using a sodium nitrite standard curve. - Concentration, the inhibition rate is calculated using the following formula:

[0104] Inhibition rate (%) = ([NO2] - ] CSE -[NO2 - ] CSE+待测样品 ) / ([NO2 - ] CSE -[NO2 - ] Control )×100%

[0105] 3) Inflammatory mediator expression inhibition assay: RAW 264.7 and THP-1 macrophages were inoculated at 5×10⁻⁶ cells / year. 5 RAW 264.7 and THP-1 cells were seeded at a density of cells / well in 12-well plates and incubated overnight at 37°C in 5% CO2 medium. RAW 264.7 and THP-1 cells were then treated with the test samples and stimulated with CSE according to the cell model construction method described above. Total RNA was extracted from RAW 264.7 and THP-1 cells using the SteadyPure kit and analyzed using NanoDrop. TM Quantitative analysis was performed using 2000 cDNA (Thermo Fisher Scientific, USA). cDNA synthesis was performed using the Evo M-MLV RT Premix kit, followed by qPCR detection using the SYBR Green Premix Pro Taq HS kit on a ViiA7 system (Applied Biosystems, USA). The qPCR program included 95.0℃ pre-denaturation for 5 minutes, followed by 40 cycles of 95.0℃ for 10 seconds and 60.0℃ for 30 seconds. Primer sequences are detailed in Table 6. Relative gene expression levels were normalized using GAPDH as an internal control and analyzed using a 2... -ΔΔCT Calculation by method.

[0106] Table 6 Primers used for PCR amplification

[0107]

[0108]

[0109] Note: * H: Human; # M: Rat.

[0110] 4) Protein Expression Inhibition Assay: RAW 264.7 and THP-1 cells were treated with test samples and stimulated with CSE according to the cell model construction method described above. Total protein was extracted from RAW 264.7 and THP-1 cells using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit, and all samples were standardized for concentration. Equal amounts of protein samples were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were then blocked with TBST solution containing 5% skim milk at 25°C for 1 hour. The blocked membranes were incubated overnight at 4°C with a 1:1000 dilution of primary antibody. After washing with TBST, the membranes were incubated with a 1:1000 dilution of goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP at room temperature for 2 hours. Finally, enhanced chemiluminescence detection was performed using an ECL kit (Merck Millipore) and analyzed by ChemiDoc. The Touch imaging system (Bio-Rad) captured protein band signals, and ImageJ software was used to perform quantitative analysis with GAPDH or β-actin (dilution ratio 1:1000) as internal controls.

[0111] All test data in steps 2)-4) are expressed as mean ± standard error, compared with the control group: ## P<0.01, ### P<0.001, #### P<0.0001; compared with the CSE group: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0112] 2. Experimental Results

[0113] (1) Cytotoxicity test results: such as Figure 5As shown in A and B, the cytotoxicity results indicate that at concentrations ≤100 μM, neither isovincinolactone nor naucleficine B had any cytotoxic effect on RAW 264.7 cells (p>0.05), and at concentrations ≤12.5 μM, neither had any cytotoxic effect on THP-1 cells (p>0.05).

[0114] (2) Results of NO generation inhibition test: such as Figure 6 As shown in the AF diagram, isoclavillarin lactone, naucleficine B, and the positive control dexamethasone (Dex) all significantly inhibited CSE-induced macrophage nitric oxide (NO) production. The IC50 values ​​of naucleficine B and isoclavillarin lactone in CSE-induced RAW 264.7 cells were [not specified in the original text]. 50 The values ​​were 0.33±0.04 and 10.70±0.82 μM, respectively, while in CSE-stimulated THP-1 cells, the IC50 values ​​of naucleficine B and isoclavella lactone were 0.33±0.04 and 10.70±0.82 μM, respectively. 50 The values ​​were 0.32±0.01 and 1.21±0.43 μM, respectively. The IC50 values ​​of Dex in inhibiting CSE-induced NO production in RAW 264.7 and THP-1 cells were [values ​​missing]. 50 The values ​​were 0.35±0.03 and 0.23±0.04 μM, respectively. This indicates that naucleficine B exhibits NO inhibitory activity comparable to dexamethasone and superior to isoclavillarin lactone.

[0115] (3) Results of inhibition of inflammatory mediator expression: such as Figure 7 As shown in the AD diagram, isovinclinolactam and naucleficine B significantly inhibited the expression of inducible nitric oxide synthase (iNOS) mRNA in a concentration-dependent manner in CSE-stimulated RAW 264.7 and THP-1 macrophages. Furthermore, Figure 8 The AD study showed that isoclavillarin lactone and naucleficine B also exhibited dose-dependent inhibitory effects on the levels of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, matrix metalloproteinases MMP-9 and MMP-12, and COX-2 in CSE-stimulated RAW 264.7 and THP-1 cells, confirming that isoclavillarin lactone and naucleficine B can inhibit the inflammatory response and achieve anti-inflammatory protective effects.

[0116] (4) Results of protein expression inhibition: such as Figures 9 to 14As shown, isoclavillarin lactone and naucleficine B downregulated the phosphorylation levels of p65, IκBα, c-Jun, c-Fos, STAT1, and STAT3 in CSE-induced RAW 264.7 and THP-1 cells, respectively, indicating that isoclavillarin lactone and naucleficine B can inhibit the activation of NF-κB, AP-1, and STAT transcription factors. Furthermore, as... Figures 15 to 18 As shown, isoclavillarin lactone and naucleficine B reduced the expression of p-JNK, p-p38, p-ERK1 / 2, TLR4 and MyD88 signaling molecules in CSE-induced RAW 264.7 and THP-1 cells, respectively, indicating that isoclavillarin lactone and naucleficine B exert anti-inflammatory effects through the MAPK and TLR4 signaling pathways.

[0117] In summary, *Gnaphalium affine* extract can improve lung function and emphysematous damage in patients with chronic obstructive pulmonary disease (COPD) by inhibiting lung inflammation, and can be used for the prevention and treatment of COPD. Furthermore, this invention is the first to isolate and identify the active components of *Gnaphalium affine* extract, including isoclavicularin lactam and naucleficine B. Naucelficine B, as a monoterpenoid indole alkaloid isolated and identified for the first time, had its molecular structure, crystal structure, and anti-inflammatory mechanism confirmed. In vitro studies have shown that isoflavone lactone and naucleficine B in *Gnaphalium affine* extract have low cytotoxicity, significantly inhibiting CSE-induced inducible nitric oxide synthase (iNOS) activity in RAW 264.7 and THP-1 cells, significantly reducing NO production induced by cigarette smoke extract (CSE), reducing the secretion of pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-8, inhibiting the expression of MMP-9, MMP-12, and COX-2 proteins, downregulating the phosphorylation levels of p65, IκBα, c-Jun, c-Fos, STAT1, and STAT3 in CSE-induced RAW 264.7 and THP-1 cells, inhibiting the activation of NF-κB, AP-1, and STAT transcription factors, and simultaneously inhibiting CSE-induced RAW 264.7 and THP-1 cell cytotoxicity. The expression of p-JNK, p-p38, p-ERK1 / 2, TLR4 and MyD88 signaling molecules in 264.7 and THP-1 cells was inhibited, and the MAPK and TLR4 signaling pathways were suppressed, thus exerting an anti-inflammatory effect.

[0118] The present invention has been further described above with reference to specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the nature and scope of the present invention. Various modifications made to the above embodiments by those skilled in the art after reading this specification are all within the scope of protection of the present invention.

Claims

1. The use of a *Gnaphalium affine* extract in the preparation of a medicament for treating chronic obstructive pulmonary disease, characterized in that... The extract of *Gynostemma pentaphyllum* was obtained by extraction using 70%-95% ethanol as a solvent.

2. The application as described in claim 1, characterized in that, The extraction method of the extract of *Gnaphalium affine* is as follows: after drying and pulverizing the trunk of *Gnaphalium affine*, add 5-10 times the mass of ethanol aqueous solution for reflux extraction. The reflux extraction time is 1-3 hours, the extraction is performed 1-3 times, and the extraction temperature is 70℃-85℃.

3. The application as described in claim 1, characterized in that, The main components of the *Cinnamomum camphora* extract include isovinctin lactam and / or the monoterpene indole alkaloid naucleficine B.

4. The application as described in claim 1, characterized in that, The drug is used to suppress lung inflammation in patients with chronic obstructive pulmonary disease.

5. The application as described in claim 1, characterized in that, The drug is used to improve lung function in patients with chronic obstructive pulmonary disease.

6. The application as described in claim 1, characterized in that, The drug is used to improve emphysematous lesions in patients with chronic obstructive pulmonary disease.

7. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that, The sole active ingredient of the pharmaceutical composition is the *Gnaphalium affine* extract as described in claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that, The content of *Gnaphalium affine* extract in the pharmaceutical composition is 1 wt% to 99 wt%.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage forms of the pharmaceutical composition include granules, tablets, capsules, oral liquids, mixtures, pills, ointments, creams, or transdermal absorption preparations.

10. The pharmaceutical composition according to claim 7, characterized in that, The drug also includes other pharmaceutically acceptable excipients; these other pharmaceutically acceptable excipients include: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

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