(-)-Epi-Osmundalactone-Rich Fraction from Angiopteris evecta Suppresses Proliferation and Induces Intrinsic Apoptosis in Non-Small Cell Lung Cancer Cells via MAPK Pathway Modulation

TL;DR

A plant extract from Angiopteris evecta shows anti-cancer effects in lung cancer cells by stopping cell growth and triggering cell death through specific molecular pathways.

Contribution

The (-)-epi-osmundalactone-rich fraction from Angiopteris evecta is newly identified as a potent anti-cancer agent targeting the MAPK pathway in NSCLC cells.

Findings

AE-EA and OLRF significantly suppressed NSCLC cell viability and clonogenic survival.

Both treatments induced G0/G1 cell cycle arrest and intrinsic apoptosis in NSCLC cells.

AE-EA and OLRF inhibited MAPK signaling by suppressing ERK1/2, JNK1/2, and p38 phosphorylation.

Abstract

Non-small cell lung cancers (NSCLCs), most notably adenocarcinoma and large cell carcinoma, have been the most frequently diagnosed lung cancer and continue to represent a leading cause of cancer-related mortality worldwide, largely due to its aggressive growth and limited therapeutic responsiveness. Natural products derived from traditional medicinal plants remain a valuable source for the discovery of novel anti-cancer agents. In this study, the anti-cancer potential of Angiopteris evecta (G. Forst.) Hoffm., a medicinal fern widely used in Thai traditional medicine, was investigated in human NSCLC, A549 and H1299 cells. Subsequential solvent extraction yielded hexane, ethyl acetate, and ethanol fractions, among which the ethyl acetate extract (AE-EA) exhibited the strongest growth inhibitory activity. Bioactivity-guided fractionation of AE-EA by thin-layer chromatography generated an (-)-epi-osmundalactone-rich fraction (OLRF), which contained three closely related lactone/furanone derivatives, with (-)-epi-osmundalactone as the predominant constituent, as determined by NMR analysis. AE-EA and OLRF significantly suppressed NSCLC cell viability and clonogenic survival and induced G0/G1 cell cycle arrest, accompanied by downregulation of cyclin D1, cyclin E1, CDK-2, and CDK-4 (p < 0.05). Both treatments also triggered intrinsic apoptosis, as evidenced by mitochondrial membrane depolarization, reduced expression of Bcl-2, and Bcl-xL, and survivin, and activation of cleaved caspase-9 and caspase-3. Mechanistically, AE-EA and OLRF significantly suppressed mitogen-activated protein kinase (MAPK) signaling through inhibition of ERK1/2, JNK1/2, and p38 phosphorylation in both NSCLC cells (p < 0.05). Collectively, these findings demonstrate that AE-EA and OLRF exert pronounced anti-cancer effects in both NSCLC cells through coordinated inhibition of MAPK signaling, induction of cell-cycle arrest, and activation of intrinsic apoptosis, supporting their potential for further development as plant-derived anti-cancer agents.

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1. Introduction

Lung cancer remains one of the leading causes of cancer-related mortality worldwide [1]. Among its subtypes, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [2]. Despite advances in molecular diagnostics and targeted therapies, the prognosis of NSCLC remains poor because of its aggressive growth, early metastatic spread, and frequent development of therapeutic resistance, particularly in advanced disease [3]. In addition to tobacco smoking, exposure to airborne particulate matter, environmental carcinogens, and genetic susceptibility all contribute to lung cancer development, emphasizing the need for more effective and broadly applicable therapeutic strategies [4].

Current treatment strategies for NSCLC are guided by tumor stage, molecular characteristics, and patient performance status. Surgical resection combined with adjuvant chemotherapy or radiotherapy is commonly applied in early-stage disease, whereas systemic therapies are the mainstay for advanced or metastatic NSCLC [5]. Although platinum-based chemotherapy, targeted therapies, and immune checkpoint inhibitors have improved outcomes in selected patients, their clinical utility is limited by toxicity, resistance, and restricted applicability to specific molecular subgroups [6,7]. These limitations highlight the urgent need for novel anti-cancer agents with improved safety profiles and multi-target activity.

At the molecular level, NSCLC is driven by genetic alterations in oncogenic pathways such as Kirsten rat sarcoma viral oncogene (KRAS), epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), mesenchymal epithelial transition factor (MET), V-Raf murine sarcoma viral oncogene homolog B (BRAF), mitogen-activated protein kinase (MAPK) or extracellular signal-related kinase (ERK) kinase (MEK) and rearranged during transfection (RET) mutations, which coverage on downstream signaling cascades, including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway (PI3K/Akt/mTOR pathway) and the rat sarcoma virus gene (RAS)/rapidly accelerated fibrosarcoma (RAF)/MAPK or ERK kinase (MEK)/ERK pathway (RAF/MEK/ERK pathway) [8,9]. Among these, aberrant activation of the MAPK pathway plays a central role in NSCLC cell proliferation, survival, and therapeutic resistance [10]. Consequently, natural products capable of modulating MAPK-dependent signaling cascades have attracted increasing interest as potential anti-cancer agents.

Natural products and phytochemicals derived from medicinal plants have historically played a central role in anti-cancer drug discovery and continue to provide structurally diverse bioactive molecules [11]. Many plant-derived compounds exert their anti-cancer effects by modulating cell cycle regulation, apoptosis, oxidative stress, and key signaling pathways [12,13,14]. Given the toxicity and resistance associated with conventional chemotherapeutics, medicinal plants remain a promising source of alternative or complementary anti-lung cancer agents.

Angiopteris evecta (G. Forst.) Hoffm. (A. evecta), a large fern of the family Marattiaceae, is commonly known as the king fern, giant fern, or elephant fern. This species is widely distributed in Southeast Asia and Oceania and has been traditionally used for the treatment of various ailments [15]. Despite its extensive ethnomedicinal use, the pharmacological activities and molecular mechanisms associated with A. evecta remain incompletely characterized. Previous studies have reported that ethyl acetate and n-butanol fractions of A. evecta rhizomes possess anti-adipogenic and anti-inflammatory activities [16], while ethanolic extracts suppress the proliferation of several human cancer cell lines, including HT-29 colon cancer and MCF-7 breast cancer cells [17,18], suggesting that this plant may possess broader biological potential.

Phytochemical analyses of A. evecta using nuclear magnetic resonance (NMR) analysis have identified multiple secondary metabolites belonging to the osmundalactone family, including angiopteroside-related derivatives [19,20]. Among these, (-)-epi-osmundalactone has been reported to exhibit anti-adipogenic and anti-inflammatory activites [16], suggesting that osmundalactone-related compounds may influence cellular processes relevant to cancer progression. However, the anti-cancer effects of A. evecta and osmundalactone-enriched fractions in lung cancer have not yet been systematically investigated.

Accordingly, the present study aimed to evaluate the anti-cancer effects of A. evecta extracts in human NSCLC cells, to identify an (-)-epi-osmundalactone-rich fraction (OLRF) derived from the ethyl acetate extract as a major bioactive component, and to elucidate the underlying molecular mechanisms. Particular emphasis was placed on assessing the regulation of cell proliferation, cell cycle progression, apoptosis, and MAPK signaling pathway modulation. Through this approach, we sought to establish A. evecta and its OLRF as promising plant-derived candidates for further development against NSCLC.

2. Results

2.1. Extraction of A. evecta and Phytochemicals Study

The crude fractional extracts were prepared from dried rhizomes of A. evecta collected from Bangkok, Thailand using n-Hex, EtOAc, and EtOH as extraction solvents. Three fractions were obtained: A. evecta ethanolic fraction (AE-EtOH), A. evecta hexane fraction (AE-Hex), and A. evecta ethyl acetate fraction (AE-EA). The EtOH extracts yielded the highest extraction efficiency (yield = 2.35%), followed by the EtOAc extracts (yield = 0.46%), whereas the n-Hex extracts produced the lowest yield (yield = 0.23%).

The phytochemical composition of A. evecta extracts was evaluated by quantifying total phenolic and total flavonoid contents. As shown in Table 1, the ethyl acetate fraction (AE-EA) exhibited the highest levels of total phenolics (90.98 ± 20.34 mg GAE/g extract) and total flavonoids (23.71 ± 7.31 mg CE/g extract), which were significantly greater than those detected in the ethanol (AE-EtOH) and hexane (AE-Hex) fractions (p < 0.001). In contrast, AE-Hex contained the lowest phenolic content, while AE-EtOH displayed intermediate levels of both phenolics and flavonoids. These findings indicate that AE-EA is enriched in polyphenolic constituents and was therefore selected for further biological and phytochemical investigations.

2.2. Identification of an (-)-Epi-Osmundalactone-Rich Fraction from the Ethyl Acetate Extract

To enrich and characterize the major bioactive constituents present in the ethyl acetate fraction of A. evecta (AE-EA), the extract was subjected to silica gel column chromatography as described in the Materials and Methods. Further fractionation was performed using preparative thin-layer chromatography (TLC). Fractions exhibiting similar TLC profiles were pooled and analyzed by ^1^H-NMR spectroscopy. One pooled TLC fraction (designed TLC fraction 4) was found to contain a mixture of three closely related lactone and furanone derivatives, among which (-)-epi-osmundalactone (5R,6R)-5,6-dihydro-5-hydroxy-6-methyl-2H-pyran-2-one) was identified as the predominant compound based on characteristic ^1^H-NMR signals and is shown in Figure 1. Two additional co-eluting constituents, (5R,6R)-5-(1-hydroxyethyl)-dihydro-2-furanone and (5R,6R)-5-(1-hydroxyethyl)-2(5H)-furanone, were also detected. This pooled subfraction was therefore defined as the (-)-epi-osmundalactone-rich fraction (OLRF) and was subsequently used for all comparative biological and mechanistic studies alongside the crude ethyl acetate extract (AE-EA).

2.3. Effects of A. evecta Extracts and OLRF on NSCLC Cell Viability

The cytotoxic effects of A. evecta extracts and the OLRF were assessed in A549 and H1299 NSCLC cells using the SRB assay. The SRB assay was selected because it provides a sensitive and reproducible measurement of cellular protein content and long-term cell viability, particularly suitable for adherent cancer cell lines and colony-forming assays. This assay can be used in an efficient and sensitive manner to test chemotherapeutic drugs or small molecules in adherent cells [21,22]. Treatment with AE-Hex and AE-EtOH resulted in minimal effects on cell viability in both cell lines following 48 h exposure (Figure 2A,B,E,F). In contrast, AE-EA markedly reduced the viability of A549 and H1299 cells in a concentration-dependent manner, exhibiting the strongest growth-inhibitory activity among all fractions, with an IC_50_ value of 67.35 ± 4.39 µg/mL in A549 cells and 52.26 ± 4.17 µg/mL in H1299 cells (Figure 2C,G).

Similarly, treatment with the OLRF significantly decreased cell viability in both A549 and H1299 cells, with an IC_50_ value of 17.14 ± 2.53 µg/mL in A549 cells and 11.56 ± 1.78 µg/mL in H1299 cells after 48 h exposure (Figure 2D,H). Regarding the safety profile in human cells, AE-EA exhibited cytotoxicity toward normal human dermal fibroblasts, with selectivity index (SI) values of 1.44 compared with A549 cells and 1.85 compared with H1299 cells (Figure S1A). OLRF also exhibited cytotoxicity toward normal human dermal fibroblasts, with SI values of 2.37 compared with A549 cells and 3.51 compared with H1299 cells (Figure S1B). Moreover, AE-EA and OLRF did not induce hemolysis of red blood cells after incubation with the extract or active compound for 4 h (Figure S2). Based on these results, AE-EA (0–40 µg/mL) and the OLRF (0–10 µg/mL) were selected for subsequent mechanistic studies.

2.4. Effects of AE-EA and OLRF on Colony Formation in NSCLC Cells

To evaluate the long-term proliferative capacity of NSCLC cells, colony formation assays were performed. AE-EA treatment significantly reduced colony formation in both A549 and H1299 cells in a dose-dependent manner (p < 0.05) (Figure 3A,B). Likewise, exposure to the OLRF fraction markedly suppressed clonogenic survival in both NSCLC cell lines (p < 0.05) (Figure 3C,D). These results demonstrate that both AE-EA and OLRF effectively impair the long-term proliferative potential of NSCLC cells.

2.5. Induction of G0/G1 Cell Cycle Arrest by AE-EA and OLRF

Flow cytometric analysis was conducted to determine whether growth inhibition was associated with alterations in cell cycle progression. Treatment with AE-EA resulted in a significant, concentration-dependent increase in the proportion of A549 and H1299 cells in the G0/G1 phase, accompanied by a reduction in S and G2/M phase populations (p < 0.001) (Figure 4A,B). A similar pattern was observed following treatment with OLRF, which also induced pronounced G0/G1 phase accumulation in both NSCLC cell lines (Figure 4C,D). These findings indicate that AE-EA and OLRF suppress NSCLC cell proliferation by arresting cell cycle progression at the G0/G1 checkpoint.

2.6. Regulation of G1/S Cell Cycle-Associated Proteins of AE-EA and OLRF

Given the observed G0/G1 cell cycle arrest, the expression of key G1/S regulatory proteins was examined by Western blot analysis. AE-EA treatment significantly reduced the protein levels of cyclin D1, cyclin E1, CDK2, and CDK4 in both A549 and H1299 cells in a concentration-dependent manner (p < 0.05) (Figure 5A,B). Consistently, treatment with the OLRF resulted in a marked downregulation of these cell cycle regulators in both NSCLC cell lines (Figure 5C,D). These results suggest that inhibition of cyclin-CDK complex formation contributes to the G0/G1 cell cycle arrest induced by AE-EA and OLRF.

2.7. Induction of Apoptosis by AE-EA and OLRF

The ability of AE-EA and OLRF to induce apoptosis was assessed using Annexin V-FITC/PI staining. Both treatments significantly increased apoptotic cell populations in A549 and H1299 cells in a dose-dependent manner (p < 0.05) (Figure 6A–D). These findings indicate that apoptosis contributes substantially to the cytotoxic effects observed in NSCLC cells following exposure to AE-EA and OLRF.

2.8. Activation of Intrinsic Apoptosis Through Mitochondrial Dysfunction by AE-EA and OLRF

Mitochondrial membrane potential (ΔΨm) analysis revealed that AE-EA treatment significantly increased the proportion of cells exhibiting mitochondrial depolarization in both A549 and H1299 cell lines (p < 0.05) (Figure 7A,B). A comparable loss of ΔΨm was observed following treatment with OLRF (Figure 7C,D).

Consistent with mitochondrial dysfunction, Western blot analysis demonstrated that both AE-EA and the OLRF decreased the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin while promoting the activation of cleaved caspase-9 and caspase-3 in both NSCLC cell lines (p < 0.05) (Figure 8A–D). These results indicate that AE-EA and OLRF induce apoptosis predominantly through the intrinsic mitochondrial pathway.

2.9. Inhibition of MAPK Signaling by AE-EA and OLRF

To investigate upstream signaling mechanisms, the effects of AE-EA and OLRF on MAPK pathway activation were examined. AE-EA treatment significantly reduced the phosphorylation levels of ERK, JNK, and p38 in both A549 and H1299 cells in a concentration-dependent manner (p < 0.05) (Figure 9A,B). Similarly, treatment with OLRF markedly suppressed ERK, JNK, and p38 phosphorylation in both NSCLC cell lines (Figure 9C,D). These findings suggest that inhibition of MAPK signaling contributes to the anti-proliferative and pro-apoptotic effects of AE-EA and OLRF.

3. Discussion

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide, largely due to its aggressive biological behavior, high metastatic potential, and frequent development of resistance to conventional therapies [23,24]. Although targeted therapies and immunotherapies have improved outcomes in selected patient subgroups, their clinical benefits remain limited by molecular heterogeneity and treatment-associated toxicities [25,26]. Consequently, the identification of novel therapeutic agents with multi-target activity and improved safety profile continues to be an important research priority. In this context, medicinal plants represent a valuable reservoir of structurally diverse bioactive compounds with potent anticancer applications [27,28]. Currently, identification of targetable mutation from tissue biopsy is generally required before applying chemotherapeutic approaches in lung cancer [29]. Many aberrant signaling cascades are implicated in the pathogenesis of lung cancer, including those involved in apoptosis pathways, growth inhibition (P53 tumor suppressor protein and serine/threonine kinase 11), and growth promotion (EGFR, cell cycle pathway, PI3K, mTOR, and MAPK) [10,29,30]. Accordingly, these pathways and their signaling molecules have become promising targets for chemotherapeutic agents.

In this study, we identified A. evecta as a promising natural source of anti-cancer activity against NSCLC. Among the tested extracts, the ethyl acetate fraction (AE-EA) exhibited the strongest cytotoxicity against A549 and H1299 cells, prompting further phytochemical investigation. Bioactivity-guided fractionation led to the identification of a TLC-derived (-)-epi-osmundalactone rich fraction (OLRF) that contained three closely related lactone/furanone derivatives, with (-)-epi-osmundalactone as the predominant constituent. Importantly, although this fraction was not chemically homogeneous, it demonstrated strong anti-proliferative and pro-apoptotic activity in both NSCLC cell lines, indicating that this chemical family represents the principal bioactive component of AE-EA. Moreover, AE-EA and OLRF demonstrated reduced cytotoxicity toward normal human fibroblasts, with an approximate two-fold selectivity index, as well as safety toward red blood cells, suggesting a degree of tumor selectivity. While preliminary, these findings support the potential therapeutic relevance of A. evecta–derived fractions and warrant further in vivo safety evaluation (Figure S1).

Previous studies have reported diverse biological activities of A. evecta, including antioxidant, antibacterial, antifungal, antihyperglycemic, and analgesic properties [18,31,32]. Ethyl acetate extracts from the leaves [18,33] and bark [18] of Angiopteris spp., including A. evecta and A. angustifolia, were shown to possess potent cytotoxicity against breast cancer cell lines (MCF-7 and MDA-MB-231), while ethanolic extracts inhibited proliferation of HT-29 colon cancer cells via induction of DNA damage and cell cycle perturbation [17]. The present study provides the first evidence that the A. evecta ethyl acetate fraction (AE-EA) and its (-)-epi-osmundalactone-rich fraction (OLRF) exert potent anti-cancer activity against NSCLC cells, thereby expanding the pharmacological profile of this medicinal fern into lung cancer. Interestingly, while Yang et al. [34] reported that osmundacetone modulates mitochondrial metabolism in A549 cells, resulting in the suppression of tumor development and cell proliferation via downregulation of GLUD1 to inhibit the glutamine/glutamate/α-KG metabolic axis and oxidative phosphorylation mechanisms, the compound investigated in that study is structurally distinct from (-)-epi-osmundalactone identified in the present work. Moreover, our study extends these observations by demonstrating MAPK pathway suppression, G0/G1 arrest, and intrinsic apoptosis using a plant-derived (-)-epi-osmundalactone-rich fraction in both A549 and p53-null H1299 cells, thereby providing broader mechanistic insight into NSCLC subtypes. The selection of A549 and H1299 cells enabled evaluation of AE-RA and OLRF across genetically distinct NSCLC subtypes. A549 cells represent lung adenocarcinoma with wide-type p53, whereas H1299 cells represent large-cell carcinoma with p53 deletion. The consistent anti-proliferative and pro-apoptotic effects observed in both models indicate that the activity of AE-EA and OLRF is not restricted to a specific p53 status and may therefore have broader relevance across NSCLC subtypes. These cell lines frequently used as in vitro models for studies of cell viability, apoptosis, colony formation, and the molecular mechanisms of anti-cancer properties due to their aggressiveness and differing p53 status [35,36]. Therefore, as our study focuses on the inhibition of NSCLC cells progression by A. evecta and one of its active compounds, (-)-epi-osmundalactone, these two cell lines are the best option to represent the aggressiveness of lung cancer in our investigation.

Mechanistically, AE-EA and OLRF inhibited NSCLC cell proliferation by inducing G0/G1 cell cycle arrest, accompanied by downregulation of cyclin D1, cyclin E1, CDK2, and CDK4. These regulators are essential for G1/S transition and DNA replication, and their suppression effectively halts cell cycle progression. AE-EA and OLRF induced cell cycle arrest in the G0/G1 phase and significantly decreased the expression of the cell cycle regulatory proteins cyclin D1, cyclin E1, CDK-2, and CDK-4. It is well documented that cell proliferation is regulated by several cyclins and cyclin-dependent kinases (CDKs) [37]. Herein, our study demonstrated that AE-EA and OLRF induced G0/G1 arrest in A549 and H1299 cells. The downregulation of cyclin D1, cyclin E1, CDK-2, and CDK-4 protein expression could result in the inhibition of DNA replication initiation process [38,39], thereby, inhibiting NSCLC cell proliferation via interruption of the cell cycle. Furthermore, it was found that AE-EA and OLRF possessed anti-cancer properties via regulation of the intrinsic apoptosis pathway. AE-EA and OLRF disrupted the mitochondrial membrane potential (ΔΨm) in both NSCLC cells. The loss of ΔΨm results in the release of cytochrome c to the cytosol, which in turn activates the caspase enzymes cascade, leading to induction of apoptosis [40]. In parallel, both treatments activated the intrinsic apoptotic pathway, as demonstrated by mitochondrial membrane depolarization, downregulation of survivin, Bcl-2, and Bcl-xL, and activation of caspase-9 and caspase-3 cleavage. This coordinated regulation of cell cycle arrest and apoptosis represents a highly desirable anti-cancer mechanism.

Recent research provides compelling evidence for the use of plant-based compounds, collectively known as phytochemicals, as anticancer agents. However, the main obstacle to successful cancer drug development is overcoming the mutation of cancer cells. Targeted therapy has therefore been established to provide novel therapeutic leads or complementary strategies for the management of aggressive lung cancers [41]. Although the prognosis for advanced lung adenocarcinoma is often poor, oncogene-directed molecular-targeted therapies can be an effective therapeutic strategy. One such oncogenic pathway involves mitogen-activated protein kinase (MAPK) which includes a signaling cascade important for tumor cell proliferation and survival [42]. A central finding of this study is the suppression of the MAPK signaling pathway by AE-EA and OLRF. Aberrant activation of ERK, JNK, and p38 signaling is a hallmark of NSCLC and contributes to tumor cell proliferation, survival, metastasis, and drug resistance [10,43,44,45]. Previous studies have found that the MAPK pathway is involved in promoting drug resistance in lung adenocarcinoma, as constitutive activation of MAPK signaling induces tumor resistance to chemotherapeutic drugs in cancer cell lines [46]. Thus, targeting these pathways could be a viable strategy to abrogate resistance in lung cancers. We demonstrated that both AE-EA and OLEF significantly reduced phosphorylation of ERK, JNK, and p38 in A549 and H1299 cells. MAPK inhibition provides a unifying explanation for the observed effects on cell cycle control and intrinsic apoptosis, as this pathway directly regulates cyclins, CDKs, and mitochondrial survival signaling.

Although OLRF is a co-eluting ternary mixture fraction, preliminary in silico docking analyses (currently under investigation) indicate that (-)-epi-osmundalactone exhibits stronger predicted binding affinity toward ERK1/2 and JNK than the co-eluting furanone derivatives, suggesting that it is likely the dominant contributor to MAPK modulation. At the same time, cooperative interactions among structurally related compounds may enhance or stabilize bioactivity, a common feature of plant-derived fractions that may offer advantages over single isolated agents.

Clinically approved agents such as cisplatin, paclitaxel, docetaxel, and gemcitabine have long been used as first-line or combination therapies for NSCLC [47] and are therefore commonly employed as reference compounds in preclinical in-vitro studies due to their well-characterized cytotoxic and pro-apoptotic effects. However, exploratory studies aimed at identifying bioactive phytochemicals or elucidating their underlying molecular mechanisms often prioritize comparisons with vehicle-treated controls to accurately define intrinsic bioactivity and specific signaling modulation prior to benchmarking against conventional drugs. In this context, the present study focused on characterizing the anti-proliferative and pro-apoptotic effects of AE-EA and the OLRF relative to vehicle-treated cells. This strategy enabled a direct evaluation of their biological activity and modulation of the MAPK signaling pathway in NSCLC cells. Nonetheless, the inclusion of standard chemotherapeutic agents as positive controls remains an important consideration for future investigations, particularly in studies designed to evaluate comparative efficacy, therapeutic index, or potential synergistic or additive effects in combination treatment settings.

In conclusion, this study demonstrates that A. evecta ethyl acetate extract and an (-)-epi-osmundalactone-rich fraction exert potent anti-cancer effects against NSCLC cells by suppressing MAPK signaling, inducing G0/G1 cell cycle arrest, and activating intrinsic apoptosis. These findings establish A. evecta as a promising botanical source for anti-lung cancer agents and provide a strong mechanistic foundation for future in vivo validation, pharmacokinetic studies, and exploration of combination therapies targeting MAPK-dependent oncogenic signaling in NSCLC.

4. Materials and Methods

4.1. Materials

Rhizomes of A. evecta were obtained from certified herbal suppliers in Bangkok, Thailand, in April 2020. Botanical authentication was performed independently by taxonomists from Chiang Mai University and Chandrakasem Rajabhat University, Thailand. A voucher specimen (WP6607) was prepared and deposited in the Queen Sirikit Botanic Garden Herbarium, Chiang Mai Province, Thailand, for future reference.

4.2. Reagents and Antibodies

Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were supplied by Gibco (Grand Island, NY, USA). RIPA lysis buffer, protease inhibitor cocktail, Commasie PlusTM Protein Assay Reagent, and enhanced chemiluminescent reagents were obtained from Thermo Fisher Scientific (Rockford, IL, USA). Sulforhodamine B (SRB), propidium iodide (PI), and anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V apoptosis detection kits were obtained from Bio-Legend (San Diego, CA, USA). Primary antibodies against cyclin D1, CDK-2, CDK-4, Bcl-2, Bcl-xL, survivin, cleaved caspase-3, cleaved caspase-9, as well as horseradish peroxidase-conjugated secondary antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA). Immobilon chemiluminescent HRP substrate was obtained from Merck Millipore.

4.3. Preparation of A. evecta Extracts

Air-dried rhizomes of A. evecta were pulverized into a fine powder and sequentially extracted using solvents of increasing polarity, namely n–hexane, ethyl acetate, and ethanol. For each extraction step, the plant material was immersed in solvent at room temperature with gentle agitation, followed by filtration. The filtrates were concentrated under reduced pressure to obtain the corresponding crude extracts. Dried extracts were weighed, dissolved in dimethyl sulfoxide (DMSO), and stored at −20 °C until further use. All extracts were subsequently subjected to biological evaluation in NSCLC cell models.

4.4. Preparation of the (-)-Epi-Osmundalactone-Rich Fraction (OLRF)

The ethyl acetate extract of A. evecta (5.0 g) was subjected to fractionation on a silica gel (grade 60) column. Elution was carried out using mixtures of n-hexane and ethyl acetate with gradually increasing polarity (from 100:0 to 0:100, v/v). The eluate was collected in 8 mL portions and monitored by thin-layer chromatography (TLC). Fractions exhibiting similar TLC profiles were pooled, resulting in four combined fractions (TLC fractions 1–4). As previously reported, TLC fraction 4 contained a high abundance of (-)-epi-osmundalactone [16]; therefore, this fraction was selected for further phytochemical characterization. Chemical profiling of fraction 4 was conducted using ^1^H-NMR spectroscopy (500 MHz; Bruker, Fällanden, Switzerland, or JEOL JNM-ECA, Tokyo, Japan). High-resolution electrospray ionization mass spectrometry (ESI-MS, TOF) was performed using a JEOL JMS-T100LC system (JEOL Ltd., Tokyo, Japan) to support structural characterization. The TLC-derived pooled fraction (TLC fraction 4) was found to contain a mixture of three structurally related lactone/furanone compounds, among which (-)-epi-osmundalactone was the predominant constituent. This pooled subfraction was therefore referred to as the (-)-epi-osmundalactone-rich fraction (OLRF). The OLRF was dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution (25 mg/mL) and stored at −20 °C until use.

4.5. Determination of Total Phenolic Content

The total phenolic content of A. evecta extracts was quantified using a modified Folin-Ciocalteu method [48]. Briefly, 0.4 mL of each extract solution was mixed with 0.3 mL of Folin-Ciocalteau reagent and incubated in the dark at room temperature for 3 min. Sodium carbonate solution (7.5%, w/v; 0.3 mL) was then added, and the mixture was further incubated for 30 min in the dark. Absorbance was measured at 765 nm using a UV visible spectrophotometer (UV-1800, Shimadzu, Japan). Gallic acid (GA) was used to generate a standard calibration curve, and results were expressed as milligrams of GA equivalents per gram of extract (mg GAE/g extract).

4.6. Determination of Total Flavonoid Content

Total flavonoid levels were determined using an aluminum chloride colorimetric assay [49]. Briefly, 250 µL of each extract was mixed with 125 µL of 5% NaNO_2_ solution and allowed to react for 5 min. Subsequently, 125 µL of 10% AlCl_3_ was added, followed by incubation for another 5 min. The reaction was then terminated by the addition of 1.0 mL NaOH, and absorbance was measured spectrophotometrically. Flavonoid content was calculated from a catechin standard curve and expressed as milligrams of catechin equivalents per gram of extract (mg CE/g extract).

4.7. Cell Lines and Culture Conditions

Human NSCLC cell lines A549 (CCL-185) and H1299 (CRL-5803) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Primary human dermal fibroblasts were aseptically isolated from an abdominal scar following a cesarean delivery performed in the operating room of Chiang Mai Maharaj Hospital, Chiang Mai University, Chiang Mai, Thailand. The study was conducted under the ethical approval of the Medical Research Ethics Committee, Chiang Mai University (Study code: BIO-2567-0035). Fibroblast cells were isolated using a previously described protocol [50]. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (50 IU/mL), and streptomycin (50 μg/mL). Cultures were grown at 37 °C in a humidified incubator containing 5% CO_2_. Cells were sub-cultured upon reaching approximately 70–80% confluence and used for experiments during the logarithmic growth phase.

4.8. Red Blood Cells Hemolysis Assay

The effects of AE-EA and ORLF on human red blood cells (RBCs) were assessed using a hemolysis induction assay following a previously described protocol [51]. Human blood samples were obtained from the Blood Bank Laboratory at Maharaj Hospital, Chiang Mai, Thailand. The samples were anonymized and could not be traced back to individual donors (study code: BIO-2567-0035, approved by the Medical Research Ethics Committee, Chiang Mai University). Briefly, packed RBCs were diluted in 0.86% normal saline solution (NSS) to prepare a 5% RBC suspension. A total of 300 μL of the 5% RBC suspension was incubated with different concentrations (0–40 μg/mL of AE-EA and 0–10 μg/mL of ORLF) at 37 °C for 4 h. NSS was used as the negative control, while 0.1% Triton X-100 served as the positive control. After incubation, the supernatant was collected by centrifugation at 5000 rpm for 5 min at room temperature, and hemoglobin concentrations were measured spectrophotometrically at 540 nm. The percentage hemolysis was calculated relative to the negative control.

4.9. Cell Viability Assay

Cell viability was evaluated using the sulforhodamine B (SRB) assay. A549 cells (5 × 10^3^ cells/well) and H1299 cells (3 × 10^3^ cells/well) were seeded into 96-well plates and allowed to attach overnight. Cells were then exposed to various concentrations of A. evecta extracts (0–200 μg/mL) or OLRF (0–50 μg/mL) for 24 or 48 h. After treatment, cells were fixed by addition of 10% (w/v) trichloroacetic acid and incubated at 4 °C for 1 h. Wells were rinsed with water, followed by staining with 0.054% (w/v) SRB solution for 30 min at room temperature. Unbound dye was removed with 1% acetic acid washes, and the plates were air-dried. Bound SRB was solubilized in 10 mM Tris-base (pH 10.5), and absorbance was measured at 510 nm using a microplate reader (Tecan Sunrise, Männedorf, Switzerland). Cell viability was calculated relative to untreated controls.

4.10. Colony Formation Assay

The long-term proliferative capacity of NSCLC cells was assessed by colony formation analysis. A549 and H1299 cells were plated at a density of 500 cells per well in six-well plates and incubated for 24 h before treatment with AE-EA (0–40 μg/mL) or OLRF (0–10 μg/mL). Untreated cells receiving vehicle alone (0.1% DMSO) served as the control group for all colony formation experiments. During the 7-day incubation period, culture medium containing the corresponding treatments was refreshed every 72 h. After 7 days, colonies were fixed with 6% glutaraldehyde and stained with toluidine blue. Colonies were imaged using an iBright™ CL-1500 imaging system (Thermo Fisher Scientific), and colony numbers were quantified. All assays were performed in triplicate.

4.11. Cell Cycle Analysis

Cells (1 × 10^6^) were seeded in six-well plates and synchronized by incubation in medium containing 0.5% FBS for 18 h. After treatment with AE-EA or OLRF for 24 h, cells were collected, washed with PBS, and fixed in 70% methanol at −20 °C overnight. Fixed cells were stained with propidium iodide (PI) solution (20 μg/mL) for 45 min at room temperature. DNA content was analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA), and cell-cycle distribution was calculated using CytExpert 2.0 software.

4.12. Apoptosis Analysis

Apoptosis was quantified using Annexin V-FITC/PI staining (BioLegend, San Diego, CA, USA). A549 and H1299 cells (1 × 10^6^ cells/well) were treated with AE-EA or OLRF for 48 h. Vehicle-treated cells (0.1% DMSO) were used as the control group. Cells were harvested, washed twice with cold PBS, and incubated with Annexin V-FITC and PI in binding buffer for 15 min in the dark. Fluorescence was measured by flow cytometry (Beckman Coulter), and apoptotic populations were analyzed within 30 min.

4.13. Mitochondrial Membrane Potential

Mitochondrial membrane potential (ΔΨm) was evaluated using MitoView™ 633 dye from Biotium (Biotium, Fremont, CA, USA). Vehicle-treated cells (0.1% DMSO) were used as the control group. After treatment with AE-EA, OLRF, or vehicle control for 24 h, cells were collected and incubated with 100 nM MitoView™ 633 in serum-free DMEM for 20 min at 37 °C in the dark. Cells were washed with PBS and fluorescence was recorded by flow cytometry (Ex/Em = 638/660 nm). Data were processed using CytExpert DxFLEX 2.0 software.

4.14. Western Blotting

Protein extracts were prepared by lysing treated cells in RIPA buffer followed by centrifugation. Protein concentrations were determined using a Bradford assay. Equal amounts of protein (20 μg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% BSA in TBS-Tween and incubated overnight at 4 °C with primary antibodies against cyclin-D1, cyclin-E1, CDK-2, CDK-4, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bcl-xL, and survivin. After washing, membranes were incubated with HRP-conjugated secondary antibodies and developed using enhanced chemiluminescence. β-Actin served as a loading control. Band intensities were quantified using ImageJ software v.1.140.

4.15. Statistical Analysis

All experiments were carried out in triplicate independent experiments to confirm reproducibility, and the data are presented as mean ± standard deviation (mean ± S.D.). Statistical analysis was carried out using Prism version 8.0 software from three independent experiments. The one-way ANOVA with Tukey’s test was used for analysis of data from cell viability, cell cycle, apoptosis, mitochondrial membrane potential, and western blot assays. Significant differences are denoted by alphabetical letters. Values with the same letter are not significantly different (p < 0.05). Significance was determined at * p < 0.05, ** p < 0.01, and *** p < 0.001. vs. control.