Network Pharmacology-Based Analysis Reveals the Mechanisms of the Tibetan Medicinal Plant Meconopsis quintuplinervia Against COPD and NAFLD: Insights from LC-MS/MS Profiling and Antioxidant/Anti-Inflammatory Activities

Abstract

Meconopsis quintuplinervia is traditionally used in Tibetan medicine for diseases of the lung and liver. This study investigated the antioxidant and anti-inflammatory activities of its extract (MQ extract), analyzed its chemical composition, and explored the potential therapeutic mechanisms against chronic obstructive pulmonary disease (COPD) and non-alcoholic fatty liver disease (NAFLD) using network pharmacology. MQ extract demonstrated effective scavenging of DPPH and ABTS radicals, with activity comparable to ascorbic acid and Trolox. In cellular assays, the extract dose-dependently reduced ROS levels in H₂O₂-induced B16-F10 and RAW264.7 cells and significantly inhibited NO production in LPS-stimulated RAW264.7 macrophages. Quantitative analysis showed total phenolic content of 90.54 ± 0.91 mg/g and total flavonoid content of 44.48 ± 0.43 mg/g. LC-MS/MS analysis identified taxifolin as the predominant constituent at approximately 2.39%. Network pharmacology and molecular docking studies revealed that flavonoids including catechin, isorhamnetin, kaempferol, luteolin, naringenin, nobiletin, quercetin, and taxifolin interacted with therapeutic targets for COPD and NAFLD. These compounds likely exerted effects by inhibiting NF-κB signaling, downregulating pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), and enhancing antioxidant enzyme activities (SOD), while also reducing hepatic lipid accumulation through SREBP-1 suppression. Our findings elucidated why Tibetan medicine traditionally uses M. quintuplinervia to treat pulmonary and hepatic disorders.

1. Introduction

The genus Meconopsis, belonging to the family Papaveraceae, comprises perennial herbaceous plants. This genus is rich in species, with approximately 60 known varieties recorded in the NCBI database, over 40 of which are found in the western regions of China, and more than 20 are utilized in the traditional Tibetan medicine system [1,2]. Meconopsis quintuplinervia, a representative medicinal plant of this genus, primarily grows in the cold and harsh environments at altitudes above 2800 m in the plateau regions of Tibet, Qinghai, and Gansu in China [1]. It is characterized by its unique ecology and significant economic value.

In terms of medicinal records, the classical Tibetan medical texts explicitly state that the entire plant can be used for medicinal purposes, with a wide range of clinical applications. Firstly, it is used to treat “lung-heat” cough and asthma caused by dry climate and wind-cold transforming into “heat” in plateau regions, characterized by symptoms such as coughing, purulent sputum, chest wall pain, and tachypnea. In modern medical terms, these symptom clusters correspond to pulmonary or bronchial disorders, including chronic obstructive pulmonary disease (COPD) [3,4]. Secondly, it is employed for jaundice due to damp-heat in the liver, assisting in clearing damp-heat and promoting bile drainage and jaundice regression, applicable for the adjunctive treatment of modern medical conditions such as hepatitis and non-alcoholic fatty liver disease (NAFLD) [5,6]. Additionally, it has certain auxiliary effects on headaches, dizziness, and plateau-specific diseases.

Given the traditional medicinal value of M. quintuplinervia, the modern medical field has conducted several systematic studies on its chemical composition and pharmacological activities. In terms of component analysis, multiple studies have confirmed its richness in flavonoid compounds [7,8,9]. The research led by Shang, through separation and purification using different solvent systems such as petroleum ether, ethyl acetate, and water, obtained four new acetylated flavonol diglucosides and five known flavonol glycosides, providing important evidence for the exploration of active components in this plant [7]. In the research on quality control and marker screening, Tian et al. established standardized extraction and detection methods: using 70% ethanol as the extraction solvent, the extract was obtained after heating reflux extraction for 1.5 h and then vacuum-dried at 50 °C [8]. The extract was further purified using D101 macroporous resin, and the 30%, 50%, and 70% ethanol eluates were collected and mixed. Finally, UPLC-QQQ-MS/MS technology was used to achieve rapid quantification of markers. This study identified nine compounds closely related to hepatocyte protection activity and in vivo absorption, including protopine, allocryptopine, caffeic acid, taxifolin, luteolin, quercetin, apigenin, chlorogenic acid, and isorhamnetin, which were recognized as the core quality markers of M. quintuplinervia. Moreover, the research by He et al. further expanded the understanding of its pharmacological activities: the ethanol extract of M. quintuplinervia is rich in phenolic and flavonoid compounds and has significant free radical scavenging ability [10]. Animal experimental results indicated that its ethyl acetate extract fraction has a protective effect against alcoholic liver injury, and its mechanism of action is speculated to be related to inhibiting lipid peroxidation reactions and reducing oxidative stress damage.

Based on the traditional use of M. quintuplinervia in Tibetan medicine for treating inflammation-related lung and liver diseases, this study aims to address critical gaps in existing research and provide innovative mechanistic insights. We initially validated the antioxidant capacity of M. quintuplinervia extract using cell-free biochemical assays (DPPH/ABTS radical scavenging) and further characterized its dose-dependent antioxidant activity (via ROS inhibition in H₂O₂-induced B16-F10 and RAW264.7 cells) and anti-inflammatory potential (by suppressing NO production in LPS-stimulated RAW264.7 macrophages). This study fills the gap of insufficient in vitro cellular-level validation of its dual bioactivities, as most existing studies have focused primarily on animal models or single-pathway effects [6,10]. Then, LC-MS/MS was employed to accurately identify and relatively quantify the chemical composition of the extract, with an emphasis on flavonoids (e.g., taxifolin, quercetin, luteolin) and phenolics. This bridges a critical gap in previous chemical-profiling studies, which focused solely on qualitative identification or quality-marker screening without establishing functional correlations. Furthermore, we combined network pharmacology with literature mining to systematically explore the potential multi-target and multi-pathway mechanisms underlying the effects of M. quintuplinervia in treating COPD and NAFLD. This approach led to the identification of novel targets—SREBP-1 and NF-κB—that have not been reported in previous M. quintuplinervia liver-disease studies, which focused primarily on IL-6, TNF, STAT3 and PPARA [6]. Additionally, this study bridges the gap in COPD-related research on M. quintuplinervia, extending its therapeutic scope beyond the previously reported liver-focused applications. Collectively, these efforts provide a scientific basis for the modern development and clinical application of M. quintuplinervia’s traditional medicinal value, while revealing new regulatory mechanisms and targets that advance our understanding of its pharmacological actions.

2. Materials and Methods

2.1. Preparation of M. quintuplinervia Extract

The plant M. quintuplinervia was identified and collected by Tamdrin Tsering, a scientist from the Science and Technology Bureau of Nyingchi, Tibet, China. A specimen was deposited at the School of Public Health and Medical Technology, Xiamen Medical College. The whole plant of the Tibetan medicine M. quintuplinervia was ground into a powder using a grinding instrument. Fifty grams of the ground powder was mixed with 300 mL of 75% ethanol, and then it was soaked at room temperature for 1 h. The soaked powder and the soaking liquid were placed into an ultrasonic extractor, and the device was started with the following parameters: “Total time”: 90 min; on for 10 s, off for 30 s, and power set at 85% (JY92-IIN sonicator, Ningbo Scientz Biotechnology, Ningbo, China). Afterward, the residue was filtered out to obtain the extract of M. quintuplinervia. The extract was then placed into a rotary evaporator for evaporation, with the water bath temperature maintained at 55 °C. Finally, the concentrated extract was freeze-dried to yield the powder of M. quintuplinervia extract (MQ extract). This extract was dissolved in 75% ethanol for subsequent experimental analysis.

2.2. Antioxidant Capacity Analysis

To analyze the antioxidant capacity, the optimal concentration of DPPH (2,2-diphenyl-1-picrylhydrazyl) working solution was prepared such that its absorbance (OD) at 517 nm was approximately 0.8 to 1.0. In a 96-well plate, 180 μL of the DPPH working solution was added to each well. Subsequently, 20 μL of M. quintuplinervia extract at different concentrations was added to achieve final concentrations of 25, 50, 100, and 200 μg/mL. The plate was then incubated in the dark for 5 min before measuring the absorbance at 517 nm. Ascorbic acid was used as the positive control group. The DPPH scavenging rate was calculated using the formula: $\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\displaystyle \raisebox{1ex}{$A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-A_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}$}\!\left/ \!\raisebox{-1ex}{$A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$}\right.}\times 100\%$. Here, A_control represents the absorbance of the DPPH solution with 75% ethanol, while A_sample represents the absorbance of the DPPH solution after adding the sample.

The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging capacity of M. quintuplinervia extract was evaluated using the T-AOC Assay Kit (ABTS) (Beyotime Biotechnology, Shanghai, China). The ABTS working solution was prepared with an absorbance at 734 nm of approximately 0.6. In a 96-well plate, 180 µL of the ABTS working solution and 20 µL of M. quintuplinervia extract at various concentrations were added to achieve final concentrations of 25, 50, 100, and 200 µg/mL. After incubating the plate in the dark for 5 min, the absorbance was measured at 734 nm. Trolox was used as the positive control. The ABTS scavenging rate was calculated using the following formula: $\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\displaystyle \raisebox{1ex}{$A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-A_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}$}\!\left/ \!\raisebox{-1ex}{$A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$}\right.}\times 100\%$. A_control represents the absorbance of the ABTS solution with 75% ethanol, while A_sample represents the absorbance of the ABTS solution after adding the sample.

The reducing power of M. quintuplinervia extract was analyzed using the T-AOC Assay Kit (FRAP) (Beyotime Biotechnology). FRAP working solution and standard solutions of FeSO₄·7H₂O at different concentrations were prepared. In a 96-well plate, 180 µL of the FRAP working solution and 20 µL of FeSO₄·7H₂O standards at various concentrations were added to each well. After incubation at 37 °C for 5 min, the absorbance was measured at 593 nm to construct a standard curve for FeSO₄.

2.3. MTT Assay for Cell Viability

All cell lines were purchased from the established commercial source BeNa Culture Collection (BNCC, Xinyang City, Henan, China). RAW 264.7 (BNCC354753) and B16-F10 cells (BNCC100309) were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C under 5% CO₂. Upon reaching ≥80% confluence, the cells were detached with trypsin, resuspended at 1 × 10⁵ cells/mL, and seeded into 96-well plates for overnight incubation. After the cells adhered to the plates, various concentrations of M. quintuplinervia extract solution were added to each well. The plates were then incubated overnight for 24 h in a 37 °C CO₂ incubator. Subsequently, 10 µL of 0.5% MTT solution was added to each well and incubated for 4 h. All the supernatant was removed, and 200 µL of DMSO was added to each well, followed by thorough mixing. The absorbance was measured at 490 nm to calculate the cell viability. The cell viability was determined using the following formula: $\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \raisebox{1ex}{$A_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}$}\!\left/ \!\raisebox{-1ex}{$A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$}\right.}\times 100\%$. A_sample is the absorbance of the wells containing the extract, and A_control is the absorbance of the wells with 75% ethanol.

2.4. Intracellular ROS Analysis

After RAW 264.7 and B16-F10 cells had adhered to the culture plates, they were treated with different concentrations of M. quintuplinervia extract and DMEM medium for 12 h. Subsequently, 400 µM H₂O₂ was added to induce a reaction for 4 h. Once the cell induction was complete, the supernatant from each well was removed. DCFH-DA (2′,7′-dichlorofluorescein diacetate) probe solution (Beyotime Biotechnology) was diluted 1:1000 in serum-free DMEM medium. An appropriate volume of the diluted probe solution, sufficient to cover the cells, was added to each well and incubated in the cell culture incubator for 20 min. The 96-well plate was gently agitated every 5 min to ensure that the probe fully penetrated the cells. After incubation, the cells were washed consecutively with serum-free cell culture medium to remove any extracellular DCFH-DA. The cells were then resuspended in 100 µL of PBS solution. Fluorescence intensity was measured in real-time using an excitation wavelength of 488 nm and an emission wavelength of 525 nm (Molecular Devices, Sunnyvale, CA, USA) for both cell types. The reactive oxygen species (ROS) fluorescence quenching rate was calculated based on the measured fluorescence intensity.

2.5. Anti-Inflammatory Activity

To assess the anti-inflammatory activity, RAW 264.7 cells were treated with different concentrations of M. quintuplinervia extract for 1 h, followed by the addition of 2 µg/mL lipopolysaccharide (LPS) and further incubation for 24 h. After incubation, the supernatant from each well was mixed with 50 µL of Griess Reagent I and 50 µL of Griess Reagent II (Nitric Oxide Assay Kit, Beyotime Biotechnology). The mixture was incubated at room temperature in the dark for 10 min. The absorbance was then measured at 540 nm. A standard curve was constructed using NaNO₂ to determine the concentration of NO in each sample.

2.6. Analysis of Total Phenolic and Total Flavonoid Content

For the analysis of total phenolic content, solutions of gallic acid at different concentrations were prepared. An aliquot of each solution was mixed with Folin-Ciocalteau reagent, vortexed to ensure homogeneity, and allowed to stand for 5 min. Subsequently, 360 µL of 20% Na₂CO₃ was added to each mixture, which was then incubated in the dark for 10 min. After incubation, the samples were centrifuged, and the supernatants were transferred to a 96-well plate. The absorbance was measured at 735 nm to construct a standard curve using gallic acid. The total phenolic content of M. quintuplinervia extract was analyzed using the same procedure, and the results were expressed as gallic acid equivalents. For the analysis of total flavonoid content, solutions of rutin at different concentrations were prepared in methanol. An aliquot of each solution was mixed with 10% AlCl₃, 1 M potassium acetate, and ddH₂O. The mixtures were allowed to react at room temperature for 30 min. The absorbance was then measured at 415 nm to construct a standard curve using rutin. The total flavonoid content of M. quintuplinervia extract was analyzed using the same procedure, and the results were expressed as rutin equivalents.

2.7. LC-MS/MS Analysis

The LC-MS/MS analysis of the MQ extract was conducted by Azenta Life Science in Suzhou, China. For sample preparation, 600 µL of methanol containing 2-chloro-L-phenylalanine (4 ppm) in MQ extract was added to the sample. Next, 100 mg of glass beads were introduced into the mixture, which was then placed in a tissue grinder and ground at 60 Hz for 90 s. The sample was subsequently subjected to ultrasonication at room temperature for 15 min, followed by centrifugation at 4 °C for 10 min. The supernatant was filtered through a 0.22 µm membrane and the filtrate was transferred to a vial for LC-MS/MS analysis. The LC analysis was performed using a Vanquish UHPLC System (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC^® HSS T3 column (150 × 2.1 mm, 1.8 µm) (Waters, Milford, MA, USA). The column temperature was maintained at 40 °C, and 2 µL of the sample was injected. The flow rate was set at 0.25 mL/min. The LC-ESI (+)-MS analysis was conducted using a mobile phase consisting of 0.1% formic acid in acetonitrile (v/v) (A) and 0.1% formic acid in water (v/v) (B). The gradient elution conditions were as follows: 0–1 min, 2% A; 1–9 min, 2–50% A; 9–12 min, 50–98% A; 12–13.5 min, 98% A; 13.5–14 min, 98–2% A; 14–20 min, 2% A. For LC-ESI (-)-MS analysis, the mobile phase consisted of (C) acetonitrile and (D) 5 mM ammonium formate. The gradient elution conditions were: 0–1 min, 2% C; 1–9 min, 2–50% C; 9–12 min, 50–98% C; 12–13.5 min, 98% C; 13.5–14 min, 98–2% C; 14–17 min, 2% C. The mass spectrometry analysis was performed using a Q Exactive Focus (Thermo Fisher Scientific, USA) with an ESI ion source. Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition was employed. The parameters were set as follows: sheath gas pressure, 30 arb; capillary temperature, 325 °C; auxiliary gas flow, 10 arb; spray voltage, 3.50 kV and −2.50 kV for ESI(+) and ESI(-), respectively; MS1 range, m/z 100–1000; MS1 resolving power, 70,000 FWHM; number of data-dependent scans per cycle, 3; MS/MS resolving power, 17,500 FWHM; normalized collision energy, 30 eV; dynamic exclusion time, automatic.

2.8. Network Pharmacology Analysis

The compounds identified in the extract of M. quintuplinervia by LC-MS/MS were subjected to a series of screening processes using the following databases: SwissADME (http://www.swissadme.ch/) (accessed on 7 August 2025) [11], TCMSP (https://www.tcmsp-e.com/tcmsp.php) (accessed on 7 August 2025) [12], SEA (https://sea.bkslab.org/) (accessed on 7 August 2025) [13], and TargetNet (http://targetnet.scbdd.com/home/index/) (accessed on 7 August 2025) [14]. In the SwissADME database, the screening criteria were set as follows: GI absorption should be “High,” and at least three “yes” responses in the Druglikeness category. In the TCMSP database, the criteria were an OB (%) value greater than 30% and a DL value greater than 0.18. For the SEA database, the criterion was a Max TC value greater than 0.1. In the TargetNet database, the criterion was a probability value greater than 0.5. The compounds that passed these screening criteria were then analyzed using the SwissTargetPrediction database to predict their potential targets. The keywords “Chronic obstructive pulmonary disease” and “non-alcoholic fatty liver disease” were used to search for relevant targets in the GeneCards (https://www.genecards.org/) (accessed on 7 August 2025) [15], OMIM (http://omim.org) (accessed on 7 August 2025) [16], and TTD (https://db.idrblab.net/ttd/) [17] databases (accessed on 7 August 2025). The intersection of these targets with those predicted for the compounds in M. quintuplinervia was analyzed to identify the potential targets for the treatment of COPD and NAFLD. The identified potential targets were then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using R software (version 4.4.1). Additionally, a “component-target-disease” network and protein–protein interaction (PPI) network for M. quintuplinervia against COPD and NAFLD were constructed using Cytoscape v3.10.3. Finally, molecular docking was performed using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/index.php) (accessed on 9 August 2025) [18] to further investigate the interactions between the compounds and their predicted targets.

2.9. Statistical Analysis

The mean values and standard deviations are presented in the figures. To analyze the significant differences among the groups, Duncan’s multiple range test was performed at the 95% confidence level using the IBM SPSS Statistics v31 software package (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Antioxidant Capacity of MQ Extract

Based on the analysis of DPPH scavenging activity, the results indicate that M. quintuplinervia (MQ) extract exhibited excellent DPPH scavenging effects (Figure 1). At a concentration of 25 μg/mL, the scavenging rate reached 54%, and at 50 μg/mL, the scavenging rate exceeded 80%. Under equivalent concentrations, its scavenging effect was superior to that of ascorbic acid. In the ABTS scavenging analysis, the performance of MQ extract was even better than that in the DPPH assay. At a concentration of 25 μg/mL, the scavenging rate surpasses 90%, and with increasing concentration, there was a slight decrease in scavenging rate due to the influence of sample coloration. This result was also better than that of the positive control group, Trolox, which required a concentration of 120 μg/mL to achieve an ABTS scavenging rate of over 90%. Furthermore, using the FRAP method to analyze the ability of MQ extract to reduce ferric-tripyridyltriazine (Fe³⁺-TPTZ) to ferrous-tripyridyltriazine (Fe²⁺-TPTZ), the results show that the reducing power of MQ extract was dose-dependent. With FeSO₄ as the standard curve, at concentrations of 125 μg/mL and 250 μg/mL, the MQ extract corresponded to FeSO₄ concentrations of 0.47 mM and 0.92 mM, respectively. The total antioxidant capacity was approximately 3.7 mM.

3.2. ROS Scavenging by MQ Extract

Given the excellent antioxidant properties of MQ extract, further investigations were conducted to explore its ability to scavenge intracellular ROS using B16-F10 and RAW264.7 cells. Prior to evaluating the ROS scavenging effects, a cell viability analysis of MQ extract was performed. The results indicated that MQ extract had no cytotoxic effects on B16-F10 cells (Figure 2). In fact, as the concentration of MQ extract increased, there was a slight trend towards increased cell viability. For RAW264.7 cells, cell viability exceeded 96% at concentrations below 100 µg/mL. However, at a concentration of 200 µg/mL, cell viability decreased to 66%, demonstrating cytotoxicity at higher concentrations.

To assess the ROS scavenging effects of MQ extract, oxidative stress in the cells was induced using H₂O₂. The results showed that the addition of H₂O₂ increased intracellular ROS levels by approximately 2.4-fold in both B16-F10 and RAW264.7 cells (Figure 2). When MQ extract was introduced, a significant decrease in intracellular ROS was observed. In B16-F10 cells, at a concentration of 200 µg/mL, intracellular ROS levels returned to those observed in the absence of H₂O₂. In RAW264.7 cells, this effect was achieved at a concentration of 50 µg/mL. These findings demonstrate that MQ extract effectively scavenges intracellular ROS.

3.3. NO Scavenging by MQ Extract

During the inflammatory process, the expression of inducible nitric oxide synthase (iNOS) is induced by pro-inflammatory cytokines (such as TNF-α and IL-1β) through the activation of transcription factors like nuclear factor-κB (NF-κB), leading to the substantial production of nitric oxide (NO) [19]. Therefore, effectively reducing NO production can indicate an effective anti-inflammatory effect. Using RAW264.7 cells for evaluation, since high concentrations of M. quintuplinervia extract are cytotoxic to RAW264.7 cells, concentrations ranging from 0 to 100 µg/mL were used for analysis. The results showed that when LPS was added, the NO content in the cells increased by approximately 1.7-fold (Figure 3). When MQ extract was added, the NO content decreased in a dose-dependent manner. At a concentration of 25 µg/mL, the NO content in the cells returned to the level observed in the absence of LPS. This indicates that MQ extract has anti-inflammatory properties.

3.4. Total Phenolic and Total Flavonoid Content of MQ Extract

Given that M. quintuplinervia contains a substantial amount of polyphenolic compounds, the total phenolic and total flavonoid contents in the MQ extract were analyzed using the Folin-Ciocalteau and AlCl₃ methods, with gallic acid and rutin serving as standards for the respective calibration curves. The results indicated that the total phenolic content in the MQ extract was 90.54 ± 0.91 mg/g-MQ extract, expressed as gallic acid equivalents. The total flavonoid content in the MQ extract was 44.48 ± 0.43 mg/g-MQ extract, expressed as rutin equivalents.

3.5. LC-MS/MS Analysis of MQ Extract

Using LC-MS/MS to analyze the components of the M. quintuplinervia extract, a total of 417 compounds were identified by mass spectrometry analysis. According to their classification, Carboxylic acids and derivatives were the most abundant (58 compounds), followed by Benzene and substituted derivatives (55), Fatty Acyls (45), Organooxygen compounds (43), Flavonoids (16), and Phenols (13) (Figure 4). Among the top 20 compounds by relative abundance, a variety of amino acids and their derivatives were most prevalent, such as L-phenylalanine, L-isoleucine, L-glutamic acid, L-tyrosine, 5-aminopentanoic acid, pyroglutamic acid, aminoadipic acid, and threonic acid (

Table 1. Top-20 most abundant metabolites identified by LC-MS/MS in M. quintuplinervia (MQ) extract.

No.	Name	mz	RT	Class	Relative Abundance	Relative Percentage
1	L-Phenylalanine	166.0852	302.4	Carboxylic acids and derivatives	3.37 × 1010 ± 3.24 × 108	11.65
2	L-Isoleucine	132.1015	191.2	Carboxylic acids and derivatives	2.70 × 1010 ± 1.56 × 1010	9.35
3	L-Glutamic acid	130.0491	156.6	Carboxylic acids and derivatives	2.12 × 1010 ± 2.49 × 108	7.32
4	4-Hydroxycinnamoylagmatine	276.1436	243.6	Cinnamic acids and derivatives	1.66 × 1010 ± 6.66 × 108	5.74
5	(6Z)-Octadecenoic acid	281.2477	827.6	—	1.51 × 1010 ± 1.22 × 1010	5.23
6	L-Tyrosine	182.0806	185.8	Carboxylic acids and derivatives	1.02 × 1010 ± 2.14 × 108	3.51
7	Hypoxanthine	137.0453	146.9	Imidazopyrimidines	8.43 × 109 ± 7.72 × 107	2.91
8	5-Aminopentanoic acid	118.0854	104.5	Carboxylic acids and derivatives	7.77 × 109 ± 2.34 × 108	2.68
9	Pyroglutamic acid	128.0356	89.7	Carboxylic acids and derivatives	7.44 × 109 ± 6.03 × 108	2.57
10	16-Hydroxy hexadecanoic acid	271.228	810.6	Fatty Acyls	7.29 × 109 ± 2.18 × 109	2.52
11	Taxifolin	303.0504	488.6	—	6.92 × 109 ± 1.65 × 108	2.39
12	D-Galactose	179.0558	91.8	—	6.67 × 109 ± 5.82 × 108	2.30
13	Aminoadipic acid	144.0648	274.1	Carboxylic acids and derivatives	6.17 × 109 ± 1.11 × 108	2.13
14	9-OxoODE	293.2125	818.9	Fatty Acyls	6.08 × 109 ± 3.73 × 109	2.10
15	Threonic acid	135.029	82.4	Organooxygen compounds	5.01 × 109 ± 1.13 × 108	1.73
16	Citric acid	173.0082	76.7	Carboxylic acids and derivatives	4.74 × 109 ± 3.25 × 108	1.64
17	13S-hydroxyoctadecadienoic acid	279.2314	813.7	Fatty Acyls	4.65 × 109 ± 1.82 × 108	1.61
18	(R)-Salsolinol	180.1012	280.6	Tetrahydroisoquinolines	4.21 × 109 ± 1.56 × 109	1.46
19	3-Hydroxymethylglutaric acid	145.0493	283.6	Fatty Acyls	3.17 × 109 ± 1.71 × 109	1.10
20	13-OxoODE	294.2153	810.8	Fatty Acyls	2.68 × 109 ± 8.33 × 108	0.92

). Additionally, the list included fatty acids, organic acids, and flavonoid compounds. Notably, Taxifolin, which accounted for approximately 2.39%, is a flavonoid known for its antioxidant and anti-inflammatory properties. These identified polyphenolic and flavonoid components corroborated the results obtained from the analysis using the Folin-Ciocalteau and AlCl₃ methods.

3.6. Network Pharmacology Analysis of MQ Extract

A total of 417 compounds identified were screened using SwissADME, TCMSP, SEA, and Targetnet databases to select those with bioavailability, drug-likeness, therapeutic potential, and high interaction with targets, resulting in 15 compounds. These include azaleatin, catechin, eriodictyol, estrone, glutaric acid, isocorypalmine, isorhamnetin, kaempferide, kaempferol, luteolin, naringenin, nobiletin, pentahydroxyflavanone, quercetin, and taxifolin (Figure 5) (

Table 2. Fifteen drug-like and target-affluent secondary metabolites of M. quintuplinervia (MQ) extract selected via SwissADME, TCMSP, SEA, and TargetNet filtering.

No.	Name	mz	RT	Class	Relative Abundance	Relative Percentage
1	Azaleatin	317.0652	441.2	Benzene and substituted derivatives	3.07 × 106 ± 9.84 × 105	0.001
2	Catechin	289.0714	414.1	Flavonoids	2.08 × 107 ± 2.79 × 106	0.007
3	Eriodictyol	287.0559	579.3	Flavonoids	1.26 × 109 ± 8.03 × 107	0.434
4	Estrone	271.1684	776.1	Steroids and steroid derivatives	1.00 × 108 ± 4.21 × 106	0.035
5	Glutaric acid	131.0342	79.2	Carboxylic acids and derivatives	3.68 × 106 ± 1.24 × 106	0.001
6	Isocorypalmine	341.1372	556.5	—	5.01 × 107 ± 1.94 × 106	0.017
7	Isorhamnetin	315.0504	657.6	Flavonoids	1.76 × 108 ± 1.14 × 107	0.061
8	Kaempferide	300.059	647.5	Flavonoids	5.98 × 108 ± 4.23 × 107	0.207
9	Kaempferol	287.0546	647.3	Flavonoids	8.01 × 107 ± 2.31 × 106	0.028
10	Luteolin	286.0434	646.9	Flavonoids	6.03 × 107 ± 4.02 × 106	0.021
11	Naringenin	271.0608	632.3	Flavonoids	6.02 × 108 ± 4.76 × 107	0.208
12	Nobiletin	403.1381	735.8	Flavonoids	3.20 × 107 ± 6.04 × 106	0.011
13	Pentahydroxyflavanone	303.0507	517.6	Benzene and substituted derivatives	4.67 × 108 ± 3.96 × 108	0.161
14	Quercetin	301.0347	615.3	Flavonoids	1.13 × 108 ± 3.97 × 107	0.039
15	Taxifolin	303.0504	488.6	Flavonoids	6.92 × 109 ± 1.65 × 108	2.392

). Most of these compounds belong to the flavonoid class, with taxifolin having the highest relative abundance, followed by eriodictyol, naringenin, kaempferide, and pentahydroxyflavanone.

A protein–protein interaction (PPI) analysis was conducted for these 15 compounds in relation to both COPD and NAFLD to identify common targets (Figure 6). For COPD, the analysis included 156 nodes, with AKT1 being the top hub target, followed by TNF, EGFR, ESR1, CASP3, HSP90AA1, PTGS2, JUN, MMP9, and NFKB1, based on degree values. For NAFLD, the analysis included 118 nodes, with AKT1 as the top hub target, followed by TNF, ESR1, HSP90AA1, JUN, NFKB1, SIRT1, CASP3, EGFR, and FOS.

A “compound-target-disease” network was constructed for the chemical components of M. quintuplinervia and the common targets of the diseases (Figure 7). For COPD, based on degree values, quercetin was the compound with the highest degree, followed by isorhamnetin, kaempferide, azaleatin, luteolin, kaempferol, glutaric acid, nobiletin, taxifolin, and eriodictyol. For NAFLD, quercetin again had the highest degree, followed by isorhamnetin, kaempferide, luteolin, kaempferol, azaleatin, glutaric acid, nobiletin, taxifolin, and eriodictyol.

GO and KEGG enrichment analyses were performed on the key targets. For GO enrichment analysis (Figure 8), the most significantly enriched genes in COPD were related to “response to xenobiotic stimulus” and “response to molecule of bacterial origin”. For NAFLD, the most significantly enriched genes were related to “response to xenobiotic stimulus” and “response to peptide hormone”. For KEGG enrichment analysis (Figure 9), COPD showed the highest GeneRatio enrichment in the “PI3K-Akt signaling pathway” and “Chemical carcinogenesis—reactive oxygen species”. NAFLD showed the highest GeneRatio enrichment in “Chemical carcinogenesis—reactive oxygen species” and “Lipid and atherosclerosis”.

Finally, molecular docking was performed using CB-Dock2, with the chemical components interacting with key targets in the “compound-target-disease” network serving as ligands. The binding energy values indicate the strength of the interaction, with lower values suggesting stronger binding affinity and higher likelihood of the active components binding to the receptor. The results, as shown in

Table 3. Top high-affinity CB-Dock2 poses (binding energy < −7.0 kcal/mol) for 14 compounds from M. quintuplinervia (MQ) extract against key therapeutic targets.

Protein	Compounds	Binding Energy (kcal/mol)	π-π	π-Cation	H-Bond	Weak H-Bond	Ionic Interaction
FOS	Isorhamnetin	−7.3	0	0	8	1	3
TLR4	Nobiletin	−8.0	2	0	1	2	1
AKT1	Quercetin	−8.2	2	0	9	6	1
TNF	Kaempferide	−8.4	0	0	6	0	0
	Isorhamnetin	−8.9	0	1	7	0	0
	Azaleatin	−9.4	0	1	8	0	0
ESR1	Taxifolin	−8.4	1	0	5	1	1
	Eriodictyol	−8.6	1	0	5	0	1
	Pentahydroxyflavanone	−8.5	1	0	5	1	1
	Naringenin	−8.4	1	0	4	2	1
	Kaempferide	−8.3	1	0	6	1	1
	Isorhamnetin	−8.3	0	0	7	1	1
	Estrone	−9.6	0	0	2	0	1
	Quercetin	−9.1	2	0	5	2	1
	Kaempferol	−8.7	2	0	4	2	1
	Luteolin	−8.8	2	0	5	2	1
	Catechin	−8.8	1	0	7	0	1
	Azaleatin	−7.7	2	0	6	5	1
HSP90AA1	Taxifolin	−9.6	1	0	4	3	0
	Eriodictyol	−9.9	2	0	8	2	0
	Pentahydroxyflavanone	−9.8	2	0	10	4	0
	Naringenin	−9.5	2	0	6	2	0
	Estrone	−10.0	2	0	1	0	0
	Catechin	−9.4	1	0	4	4	0
PTGS2	Kaempferide	−9.5	2	0	4	1	0
	Isorhamnetin	−9.2	1	0	11	3	0
	Quercetin	−10.0	2	0	6	1	0
	Kaempferol	−9.7	1	0	4	0	0
	Luteolin	−9.7	2	0	5	1	0
	Nobiletin	−8.1	2	1	2	6	0
	Azaleatin	−9.7	2	0	7	2	0
MMP9	Kaempferide	−9.5	0	0	5	3	0
	Quercetin	−9.7	0	0	8	5	0
	Luteolin	−10.1	1	0	8	5	0
GSK3B	Kaempferide	−8.0	0	0	7	6	0
	Isorhamnetin	−8.4	2	0	8	5	1
	Quercetin	−8.3	1	0	6	3	2
	Kaempferol	−8.3	0	0	11	3	0
	Luteolin	−8.3	0	0	6	3	0
	Azaleatin	−8.0	0	0	11	3	0
CREB1	Kaempferide	−7.5	0	0	4	0	1
	Isorhamnetin	−7.5	0	0	3	0	1
	Quercetin	−7.9	0	0	5	0	1
	Kaempferol	−7.1	0	0	3	0	1
	Azaleatin	−7.3	0	0	6	0	1
JUN	Isocorypalmine	−8.7	0	1	3	2	2
EGFR	Quercetin	−10.1	0	0	13	5	0
APP	Kaempferide	−8.3	0	0	13	9	0
	Isorhamnetin	−8.3	0	0	15	9	0
	Estrone	−7.8	0	0	9	6	0
	Quercetin	−8.3	0	0	14	7	0
	Kaempferol	−8.6	0	0	13	8	0
	Luteolin	−8.1	0	0	11	7	0
	Azaleatin	−7.8	0	0	7	6	0

, indicate that targets such as ESR1, HSP90AA1, PTGS2, GSK3B, CREB1, and APP have multiple compounds binding to them. Glutaric acid displayed a comparatively high binding energy, suggesting that it may not participate in the relevant mechanism.

4. Discussion

In this study, M. quintuplinervia from Tibet was extracted using 75% ethanol to comprehensively retain medium- and high-polarity bioactive components, including polyphenols and flavonoids. Our in vitro experiments confirmed that the extract exhibited potent antioxidant activity—effectively scavenging DPPH and ABTS radicals (with efficacy comparable to ascorbic acid and Trolox) and dose-dependently reducing intracellular ROS levels in H₂O₂-induced B16-F10 and RAW264.7 cells. Additionally, the extract significantly inhibited NO production in LPS-stimulated RAW264.7 macrophages, demonstrating robust anti-inflammatory potential. These bioactivities were directly attributed to the extract’s high total phenolic content (90.54 ± 0.91 mg/g, gallic acid equivalents) and total flavonoid content (44.48 ± 0.43 mg/g, rutin equivalents), which aligns with He et al.’s report that ethanol extracts of M. quintuplinervia possess strong free radical-scavenging capacity [10].

Through LC-MS/MS profiling, we identified 417 compounds in the extract, and further screening (integrating oral bioavailability, drug-likeness, and target affinity via SwissADME, TCMSP, SEA, and TargetNet databases) yielded 15 key bioactive metabolites (Table 2). Flavonoids accounted for the highest proportion of these candidates, with taxifolin exhibiting the highest relative abundance (2.39%), followed by eriodictyol, naringenin, and kaempferide. Notably, our network pharmacology and molecular docking analyses confirmed that these flavonoids (e.g., catechin, isorhamnetin, kaempferol, luteolin, quercetin, taxifolin) interact with core therapeutic targets for COPD and NAFLD—including AKT1, TNF, ESR1, HSP90AA1, and NFKB1 (Figure 6 and Figure 7). These interactions were further validated by favorable binding energies (≤ −7.0 kcal/mol) in molecular docking (Table 3), indicating stable compound-target binding.

Collectively, a key rationale for discussing each identified compound individually lies in the strong alignment between our findings and existing experimental evidence supporting their roles in pulmonary and hepatic protection. Notably, some of these identified metabolites have been previously reported to exert protective effects against COPD or NAFLD, with azaleatin and isocorypalmine being newly implicated here. Below, we systematically elucidate how each compound’s established pharmacological roles, combined with our current study’s bioactivity data and docking results, collectively underpin the plant’s therapeutic efficacy for pulmonary and hepatic inflammation-related diseases.

4.1. Azaleatin

Azaleatin, a methylated derivative of quercetin with a methoxy substitution at the C5 position, exerts a non-competitive inhibitory effect by targeting the allosteric pocket of the dengue virus NS2B-NS3 protease [20]. In addition, this compound potently suppresses glutaminyl cyclase, an enzyme closely associated with the pathogenesis of Alzheimer’s disease [21]. However, no research has yet investigated the potential efficacy of azaleatin in the treatment of COPD or NAFLD. In this study, molecular docking analyses have revealed that azaleatin exhibited robust binding affinities to multiple target proteins, including TNF, ESR1, PTGS2, GSK3B, CREB1 and APP. To date, experimental validation has only confirmed that azaleatin can significantly downregulate the sodium arsenite-induced overexpression of TNF-α, thereby blocking the TNF-α-mediated inflammatory cascade and conferring cardioprotective effects [22].

4.2. Catechin

Catechin, a natural flavonoid abundant in tea and fruits, exhibits antioxidant, anti-inflammatory, and potential estrogen-like activities. Data from the large-scale MORGEN cohort (13,651 adults) revealed a positive association between catechin intake and lung function, as well as a negative correlation with COPD symptoms. The high-intake group showed a markedly greater improvement than the low-intake group [23]. In the adjunctive treatment of NAFLD, catechin significantly reduces liver injury markers such as Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) by modulating metabolism, inhibiting oxidative stress and inflammation, and restoring gut microbiota balance [24,25,26,27]. Recent network pharmacology and molecular docking studies further indicate that catechin can form stable bindings with ESR1 and HSP90AA1. Potential “docking interactions” have been predicted in models of hepatocellular carcinoma, ulcerative colitis, and irritable bowel syndrome, providing computational evidence for its multi-target therapeutic potential [28,29].

4.3. Eriodictyol

Eriodictyol is a flavanone flavonoid widely found in medicinal plants, citrus fruits, and vegetables. A cross-sectional NHANES study (involving 11,743 U.S. adults) indicated an inverse correlation between total flavonoid intake and the risk of chronic respiratory diseases, with eriodictyol contributing most significantly to asthma risk reduction (32.13%), suggesting its potential role in improving respiratory health through anti-inflammatory and antioxidant pathways [30]. Although no clinical studies have directly reported the use of eriodictyol in NAFLD treatment, it has been demonstrated to exert anticancer and neuroprotective effects by simultaneously inhibiting oxidative stress, inflammation, and apoptosis via multi-target and multi-pathway networks at low micromolar concentrations [31]. In this study, molecular docking simulations further suggest that eriodictyol, similar to catechin, can bind to ESR1 and HSP90AA1; however, its actual binding affinity and functional activity await experimental validation.

4.4. Estrone

Estrone (E1), the most abundant estrogen in the human body, is stored as fatty acid esters in white adipose tissue and can be rapidly converted to estradiol via 17β-HSD, thereby regulating energy metabolism and glucose homeostasis through ERα/ERβ signaling [32,33]. In a multivariate analysis of 573 children with biopsy-confirmed NAFLD, higher serum estrone levels were associated with reduced portal inflammation and fibrosis, suggesting a potential protective role of estrone in pediatric NAFLD [34]. Further in vitro studies revealed that estrone significantly promotes proliferation in ERα-positive Ishikawa cells but inhibits growth in ERα-negative KLE cells, confirming that ESR1 expression is a key determinant of its estrogenic activity [35]. As a member of the neurosteroid family, estrone also enhances ATP production, respiratory chain function, and glycolytic capacity in APP/Aβ Alzheimer’s disease model cells, offering a novel therapeutic strategy for AD [36]. To date, however, no studies have explored the role of estrone in COPD, nor has its potential interaction with HSP90AA1 been experimentally validated.

4.5. Isocorypalmine

Isocorypalmine is a natural tetrahydroprotoberberine (THPB) alkaloid. It functions as a competitive antagonist of the dopamine D2 receptor (D2R), thereby exerting sedative effects [37], and shows potential for intervening in cocaine use disorder by modulating the dopaminergic system [38]. However, there are currently no research reports on its application in COPD or NAFLD, and its molecular docking with the transcription factor JUN also lacks experimental validation.

4.6. Isorhamnetin

Isorhamnetin, a methylated derivative of quercetin capable of crossing the blood-brain barrier, possesses antioxidant, anti-inflammatory, anticancer, and cardiovascular activities. It significantly alleviates lung injury by inhibiting NF-κB/HMGB1, TNF-α/IL-1β/IL-6 [39], TLR4, and mTOR while activating autophagy [40]. Isorhamnetin intervention also suppresses SREBP-1c [41] and SLCO1B3 [42], thereby improving steatosis, fibrosis, and bile acid metabolism in NASH/NAFLD. Experimental studies have confirmed its ability to bind and modulate FOS, TNF, ESR1, and CREB1. It reduces the expression of FOS (a gene involved in osteoclast function) by inhibiting the RANKL/RANK axis and downregulating NFATc1 [43], decreases TNF-α to alleviate inflammatory damage [44], downregulates ESR1 to inhibit ovarian cancer progression [45], and regulates CREB1/p-CREB to produce analgesic and neuroprotective effects [46], highlighting its potential as a multi-target candidate for the treatment of COPD and NAFLD.

4.7. Kaempferide

Kaempferide, a flavonol derivative, is widely distributed across numerous medicinal plants of the Zingiberaceae family [47]. It effectively mitigates oleic acid-induced hepatic lipid accumulation and oxidative stress by downregulating SREBP-1/SCD-1, PPARγ, and perilipin, while inhibiting the overactivation of the Nrf2/HO-1 pathway, thereby suppressing de novo fatty acid synthesis and lipid peroxidation [48]. These mechanisms support its potential as a multi-target natural candidate for NAFLD. However, its protective effects in the lung have not yet been reported. In this study, molecular docking studies indicate that kaempferide can bind to TNF, ESR1, PTGS2, MMP9, GSK3B, CREB1, and APP. To date, experimental validation has only confirmed its ability to reduce TNF-α release in LPS+ATP-induced cardiac fibroblasts via the NF-κB/Akt pathway [49], while interactions with the remaining targets await further verification.

4.8. Kaempferol

Kaempferol is a naturally occurring flavonoid belonging to the flavonol subclass, widely present in fruits, vegetables, tea, and medicinal plants. It alleviates pulmonary inflammation and lung injury induced in H9N2 influenza, LPS, bleomycin, and asthma models by modulating signaling pathways such as TLR4/MyD88/NF-κB, Syk-PLCγ, PKCμ-ERK-cPLA2-COX2, and NF-κB, thereby reducing associated symptoms [50]. Additionally, it activates Sirt1/AMPK [51], inhibits NLRP3-ASC [52], and downregulates ACC/SREBP1 [53], improving lipid deposition and inflammation in NASH/NAFLD. In this study, molecular docking suggests kaempferol can bind to targets such as ESR1, PTGS2, and CREB1. Experimentally, it has been shown to inhibit endometrial cancer through HSD17B1-associated genes such as ESR1, ESRRA, and PPARG [54], block the active site of PTGS2 via hydrogen bonding to reduce PGE2 and heterotopic ossification [55], and restore suppressed CREB phosphorylation and transcriptional activity in pancreatic β-cells under chronic high-fat conditions [56]. However, its binding to APP and GSK3B remains to be verified.

4.9. Luteolin

Luteolin is a common flavonoid compound belonging to the flavone subclass, structurally similar to kaempferol. In COPD, acute lung injury/ARDS, and pulmonary fibrosis models, luteolin reduces oxidative stress, ROS, and inflammatory responses by modulating pathways such as TRPV1/SIRT6, CYP2A13/NRF2 [57], PI3K/Akt/NF-κB, and MAPK [58]. In NAFLD/NASH, it improves insulin resistance, reduces lipid droplets, ALT, and TNF-α by regulating AMPK/PGC-1α and PI3K/Akt/FoxO1, downregulates SREBP1c and TLR4/NF-κB [59,60,61]. In this study, molecular docking and experimental studies indicate that luteolin can bind and modulate targets including ESR1, PTGS2, MMP9, APP, and GSK3B. Specifically, it upregulates ESR1 to inhibit hepatocellular carcinoma [62], downregulates PTGS2 to prevent vascular smooth muscle cell (VSMC) proliferation and inflammatory response [63], reduces MMP9 to suppress metastasis in melanoma [64], decreases p-GSK3β to promote apoptosis in cervical cancer cells [65], and reduces APP/PS1 binding to alleviate Alzheimer’s pathology [66].

4.10. Naringenin

Naringenin is a dihydroflavone compound belonging to the flavonoid family, and one of the most abundant natural active constituents in citrus fruits. It mitigates lung injury induced by LPS, cigarette smoke, and radiation through modulation of the PI3K/AKT and NF-κB pathways, reduction of ROS generation, and enhancement of SOD/CAT activity [67]. In the liver, naringenin reverses lipid accumulation and elevated ALT/AST levels in methionine–choline-deficient (MCD)-induced NASH [68], while also reducing hepatic fat content and improving lipid profiles in NAFLD patients [69]. In this study, molecular docking confirms that naringenin stably binds only to ESR1 and HSP90AA1. It downregulates ESR1 to inhibit the activity of MCF-7 breast cancer cells [70], and modulates HSP90AA1 to improve ovarian pathology and estrous cyclicity in polycystic ovary syndrome (PCOS) mice [71]. These findings provide a basis for naringenin’s potential in targeting estrogen-related pathways for the prevention and treatment of metabolic and hormone-associated disorders.

4.11. Nobiletin

Nobiletin is a polymethoxylated flavone (PMF) belonging to the flavonoid family, predominantly found in citrus plants. By modulating the NF-κB, p38 MAPK, and AKT pathways, nobiletin alleviates inflammation and oxidative stress in acute lung injury models induced by LPS, bacterial infection, and pancreatitis [72]. It also regulates the gut–liver axis by restoring the Firmicutes/Bacteroidetes ratio and Akkermansiaceae abundance, reducing serum ALT, AST, and LPS levels, repairing the intestinal barrier, and inhibiting the NF-κB/TLR4 pathway to protect against alcohol-induced liver injury [73]. In this study, molecular docking suggests that nobiletin exhibits high affinity specifically for TLR4 and PTGS2. Experimental studies have confirmed that it markedly inhibits the NF-κB/TLR4 signaling pathway in liver injury [73]. Moreover, nobiletin suppresses the activation of NF-κB and AKT1 pathways and dose-dependently reduces abnormally elevated PTGS2 expression, thereby decreasing the release of inflammatory cytokines and matrix metalloproteinases [74].

4.12. Pentahydroxyflavanone

Pentahydroxyflavanone is a highly polar flavanone characterized by five hydroxyl groups distributed across its B and A rings, commonly found in plants as either an aglycone or a glycoside. To date, no studies have reported its protective effects against lung or liver injury. In this study, molecular docking simulations have only suggested potential binding of pentahydroxyflavanone to ESR1 and HSP90AA1; however, its binding affinity and functional effects remain to be experimentally validated.

4.13. Quercetin

Quercetin is a natural flavonol belonging to the flavonoid polyphenol family, widely distributed in vegetables, fruits, tea, and medicinal plants, and is one of the most extensively studied flavonoids. In COPD models, quercetin alleviates pulmonary inflammation and fibrosis by inhibiting NLRP3/IL-1β and TGF-β1 while upregulating SOD/GSH-Px [75]. In NAFLD, it reduces hepatic steatosis and hepatocyte injury by activating autophagy markers LC3A and p62 [76], and blocking the NF-κB pathway to lower TNF-α [27]. Molecular docking and experimental studies demonstrate that quercetin can bind to and modulate multiple targets, including AKT1, ESR1, PTGS2, MMP9, GSK3B, CREB1, EGFR, and APP. Specifically, it downregulates p-AKT to promote apoptosis in non-small-cell lung cancer (NSCLC) [77]; modulates ESR1 to enhance apolipoprotein A-I (apo A-I) gene expression, thereby improving lipid metabolism [78]; inhibits PTGS2 to alleviate hyperoxia-induced bronchopulmonary dysplasia (BPD) [79]; downregulates MMP9 to suppress esophageal cancer invasion and angiogenesis [80]; activates GSK3B to exert anti-cardiac hypertrophic effects [81]; upregulates CREB1 to prolong memory [82]; blocks the AREG/EGFR axis to attenuate renal fibrosis [83]; and downregulates APP/BACE1 to ameliorate early-stage Alzheimer’s disease pathology [84].

4.14. Taxifolin

Taxifolin (also known as dihydroquercetin) is a dihydroflavonol, a type of flavonoid polyphenol and a 2,3-saturated derivative of quercetin. It is abundant in larch and various Chinese medicinal herbs [85]. In cigarette smoke-induced COPD, taxifolin alleviates emphysema and apoptosis of lung parenchymal cells by inhibiting NF-κB, downregulating IL-1β/IL-6, and modulating Bax/Bcl-2/Caspase-3 [86]. In NASH, it blocks lipid accumulation, activates FGF21-mediated energy metabolism in brown adipose tissue, suppresses pro-inflammatory and pro-fibrotic genes, and reduces hepatic lipid content, inflammation, and fibrosis, thereby delaying hepatocellular carcinoma development [87]. In this study, molecular docking simulations suggest that taxifolin may bind to ESR1 and HSP90AA1; however, its binding affinity and functional effects remain to be experimentally validated.

5. Conclusions

It is important to note that the present study represents a preliminary mechanistic exploration of M. quintuplinervia’s therapeutic potential against COPD and NAFLD, primarily based on in vitro bioassays, chemical profiling, and in silico network pharmacology and molecular docking analyses. While our findings establish a clear link between the plant’s flavonoid-rich composition and its antioxidant/anti-inflammatory activities, and predict key compound-target-pathway interactions, direct in vivo validation remains to be conducted. Flavonoids and alkaloids enriched in M. quintuplinervia form a synergistic “multi-target, multi-pathway” network. Components such as catechin, eriodictyol, isorhamnetin, kaempferol, luteolin, naringenin, nobiletin, quercetin, and taxifolin alleviate COPD-related emphysema and airway remodeling by inhibiting NF-κB, TLR4, NLRP3 inflammasome, and TGF-β1, downregulating TNF-α, IL-6, and IL-1β, enhancing SOD, CAT and GSH-Px, and suppressing cell apoptosis and ROS production. Concurrently, they block de novo fatty-acid synthesis, inhibit SREBP-1c-mediated de novo lipogenesis, and reduce hepatic lipid accumulation and oxidative stress, thereby preventing the progression of NAFLD to NASH and liver fibrosis. Estrone regulates energy metabolism via ERα, while isorhamnetin and nobiletin simultaneously modulate the gut–liver axis and bile acid homeostasis. Additional pathways—such as those involving ER, HSP90 or PTGS2—also warrant exploration as potential therapeutic avenues for future research on pulmonary and hepatic inflammation. In Tibetan medicine, this plant has traditionally been used for lung “heat syndromes” and hepatic “fat stagnation,” leveraging its broad-spectrum anti-inflammatory, antioxidant, and anti-fibrotic synergistic effects. Modern molecular docking and experimental studies have provided a molecular basis for its clinical efficacy against COPD and NAFLD. Future animal studies are warranted to further confirm and extend these preliminary insights. Priority directions will include evaluating the in vivo efficacy of M. quintuplinervia extract and its key bioactive compounds using established rodent models of COPD and NAFLD, validating the predicted molecular mechanisms by assessing changes in core targets and pathway activity in lung and liver tissues, investigating the pharmacokinetic profiles of major bioactive flavonoids, and exploring the synergistic effects of multi-component interactions. Additionally, long-term safety assessments will be conducted to support the plant’s potential development as a nutraceutical or therapeutic agent.