Dried flower heads supplied as CI were purchased from HORN GIN TRADING CO., LTD. Subsequently, 1,004.5 g dried material was pulverized and extracted with 95% (volume/volume) ethanol at room temperature for 24 h. The extraction procedure was repeated three times. The combined filtrates were concentrated under reduced pressure at ≤40°C using a rotary evaporator (60 rpm) to obtain a dark brown crude extract. After drying, 245.35 g crude extract was obtained, corresponding to an extraction yield of 23.93% (weight/weight), with a moisture content of 2.63%. The dried extract was stored at −20°C until subsequent biological experiments. Prior to cellular experiments, the dried CI extract was dissolved in absolute ethanol to prepare a 100 mg/ml stock solution and subsequently diluted in culture medium to the indicated final concentrations. These defined extraction parameters were maintained to ensure procedural consistency.

Plant material supplied as CI was subjected to macroscopic examination, including assessment of its appearance, color and odor. Microscopic analysis of tissue sections was additionally performed using Fast Green staining to visualize tissue morphology and Periodic Acid-Schiff (PAS) staining to detect polysaccharide-containing structures and characterize anatomical features. Plant tissue samples were fixed in 4% paraformaldehyde at 4°C overnight. Following fixation, the samples were washed twice with 1X PBS for 30 min each on ice, dehydrated through a graded ethanol series (10, 30, 50, 70, 85, 95 and 100%), cleared in xylene and infiltrated with molten paraffin at 60°C. The tissues were then embedded in paraffin and sectioned at a thickness of 4–8 µm using a microtome. For Fast Green staining, paraffin-embedded sections were deparaffinized twice in xylene for 5–10 min each and rehydrated through a graded ethanol series (100, 95, 80 and 70%) to distilled water. The sections were stained with 0.05% (w/v) Fast Green FCF (Sigma-Aldrich; Merck KGaA) for 1 min at room temperature. The sections were subsequently rinsed, dehydrated through a graded ethanol series, cleared in xylene and mounted using a resin-based mounting medium. For Periodic acid-Schiff (PAS) staining, deparaffinized and rehydrated sections were stained using a PAS staining kit (Muto Pure Chemicals Co., Ltd.). Briefly, the sections were oxidized with 1% periodic acid for 5 min at room temperature, rinsed thoroughly with distilled water and incubated with Schiff reagent for 10 min at room temperature. The sections were then washed under running tap water for 5 min, counterstained with hematoxylin for 30 sec, dehydrated through a graded ethanol series, cleared twice in xylene and mounted using a resin-based mounting medium. PAS-positive polysaccharide-rich structures were identified by their characteristic magenta staining. Stained sections were examined and photographed using an Eclipse 4,000 light microscope (Nikon Corporation) at ×400 magnification. Formal taxonomic identification by a qualified botanist or taxonomist was not performed, and no voucher specimen was deposited in a recognized herbarium or institutional collection.

The human HSCC cell line FaDu was used as the in vitro model in the present study. Cells were maintained in EMEM supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO₂. Upon reaching ~80% confluence, cell monolayers were detached using 0.25% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) and routinely subcultured every 2–3 days to maintain exponential growth.

To quantitatively assess changes in intracellular oxidative activity, FaDu cells were analyzed using the cell-permeable, ROS-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Cayman Chemical Company) according to established redox biology protocols (14,16). Briefly, FaDu cells were seeded in 6-cm culture dishes and allowed to adhere overnight at 37°C in a humidified atmosphere containing 5% CO₂, reaching ~80% confluence prior to treatment. Cells were subsequently treated with CI extract at concentrations of 1 or 2 mg/ml for 24 h. Vehicle-control cells were treated with an equivalent volume of absolute ethanol. For the ROS-scavenging control, a subset of cells was pre-incubated with 5 mM N-acetylcysteine (NAC; Sigma-Aldrich; Merck KGaA) for 1 h prior to CI treatment. Following the 24-h treatment period, cells were harvested using 0.5% Trypsin-EDTA without phenol red (cat. no. 15400054; Gibco; Thermo Fisher Scientific, Inc.) for 5 min at 37°C and transferred to centrifuge tubes. Cell pellets were washed twice with ice-cold PBS through centrifugation 300 × g for 5 min, resuspended and incubated with 10 µM DCFH-DA for 30 min at 37°C in the dark. Fluorescence signals were subsequently detected using a BD FACSLyric™ flow cytometer (BD Biosciences). Cellular debris was excluded through appropriate gating and a minimum of 10,000 events were acquired per sample. Intracellular oxidative activity was quantified according to the mean fluorescence intensity using FACSuite software (BD Biosciences).

The expression levels of key proteins involved in endoplasmic reticulum (ER) stress and cell survival pathways were evaluated using western blotting. Following 24 h of CI treatment, FaDu cells were harvested, washed with ice-cold PBS and lysed in RIPA lysis buffer (Sigma-Aldrich; Merck KGaA) supplemented with 1% protease and phosphatase inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min. Total protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein (30 µg per lane) were separated using 10% SDS-PAGE and transferred onto PVDF membranes (MilliporeSigma; Merck KGaA). Nonspecific binding sites were blocked with 5% BSA in TBS containing 0.1% Tween 20 (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The membranes were subsequently incubated overnight at 4°C with primary antibodies against EIF2AK3 (cat. no. 24390-1-AP; 1:1,000; Proteintech Group, Inc.), Bcl-2 (cat. no. 26593-1-AP; 1:1,000; Proteintech Group, Inc.), phosphorylated (p)-Akt (cat. no. 4060; 1:1,000; Cell Signaling Technology, Inc.), total Akt (cat. no. 4691; 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. AM4300; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.), with GAPDH used as the loading control. The membranes were washed three times with TBST for 10 min each and incubated with the appropriate HRP-conjugated secondary antibodies (Goat Anti-Mouse IgG cat. no. 115-035-003; and Goat Anti-Rabbit IgG; cat. no. 111-035-003; 1:5,000; Jackson ImmunoResearch, Inc.) for 1 h at room temperature. Following three additional washes with TBST, immunoreactive protein bands were visualized using an ECL detection kit (Revvity, Inc.) and captured using a ChemiDoc MP digital imaging system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed using ImageJ (Version 1.53; National Institutes of Health) and the relative expression levels of target proteins were normalized to the corresponding GAPDH loading controls.

The cytotoxic activity of CI extract was evaluated using a CCK-8 assay (Sigma-Aldrich; Merck KGaA). To exclude potential cytotoxic effects of the solvent, cells in the control groups for all in vitro assays were treated with an equivalent volume of absolute ethanol, with a maximum final concentration of 2% (v/v), as the vehicle control. Preliminary experiments determined that this ethanol concentration did not induce marked cytotoxicity compared with untreated cells. FaDu cells were seeded into 96-well flat-bottom plates at a density of 6×10³ cells/well and incubated overnight to allow cell attachment. The culture medium was subsequently replaced with fresh medium containing the indicated concentrations of CI extract, and cells were incubated for an additional 48 or 72 h. At the indicated time points, 10 µl CCK-8 reagent was added directly to each well, followed by incubation for 1.5 h. Absorbance at 450 nm was measured using a microplate reader (BioTek; Agilent Technologies, Inc.). Cell viability was calculated relative to the vehicle control group, which was designated as 100%. IC₅₀ values were calculated through nonlinear regression analysis of concentration-response curves using GraphPad Prism (version, 10.0; Dotmatics).

All in vitro experiments were performed using three independent biological replicates (n=3). For the CCK-8 assay, each condition was analyzed in triplicate wells within each independent experiment. Comparisons between two groups were performed using an unpaired Student's t-test, while comparisons among multiple groups were performed using ANOVA followed by Tukey's multiple comparisons test. For CCK-8 experiments involving both concentration and time as independent variables, a two-way ANOVA followed by Tukey's post hoc test was used. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To characterize the morphological and anatomical features of the raw material, dried flower heads of CI were subjected to macroscopic and microscopic evaluation. Macroscopic examination revealed a capitulum structure, comprising yellow tubular central florets and outer ray florets, together with a distinct aromatic odour, consistent with the morphological features expected for the material supplied as CI (Fig. 1A). Histological cross-sections stained with Fast Green and PAS further demonstrated the anatomical architecture of the floral tissues. PAS staining delineated polysaccharide-rich cell walls and secretory glandular trichomes in magenta, whereas Fast Green provided contrasting visualization of cytoplasmic components in green. Collectively, these findings documented the morphological and anatomical characteristics of the plant material (Fig. 1A). Following morphological and histological assessment, 1,004.5 g pulverized flower heads was extracted with 95% ethanol. Following evaporation of the solvent under reduced pressure, a dark brown, viscous crude extract was obtained (Fig. 1B). The resulting crude extract was aliquoted and stored for subsequent in vitro pharmacological and molecular experiments.

To characterize the early cellular response to CI treatment, changes in intracellular oxidative activity were evaluated using the cell-permeable fluorogenic probe, DCFH-DA. Following cellular uptake, DCFH-DA is deacetylated through intracellular esterases and subsequently oxidized to the fluorescent compound DCF. Flow cytometric analysis revealed a notable concentration-dependent rightward shift in green fluorescence in FaDu cells treated with 1 or 2 mg/ml CI for 24 h (Fig. 2A). Quantitative analysis demonstrated that the relative mean fluorescence intensity was significantly higher in CI-treated cells compared with vehicle-treated controls (Fig. 2B), indicating increased intracellular oxidative activity following CI treatment (P<0.01; Fig. 2).

To determine whether the CI-induced increase in DCF fluorescence was sensitive to antioxidant pretreatment, a rescue experiment was performed using NAC, an antioxidant and ROS scavenger. FaDu cells were pre-incubated with 5 mM NAC for 1 h before CI treatment. NAC pretreatment markedly attenuated the CI-induced increase in DCF fluorescence, reducing the fluorescence signal toward baseline levels (Fig. 2). Collectively, these findings support the involvement of increased intracellular oxidative activity in the cellular response to CI treatment.

To further characterize the molecular alterations associated with CI treatment, proteins involved in ER stress adaptation and cell survival were evaluated. FaDu cells were treated with 1 or 2 mg/ml CI for 24 h, followed by western blotting of whole-cell lysates. EIF2AK3, also known as PERK, exhibited a significant concentration-dependent reduction following CI treatment, as supported through densitometric semi-quantification normalized to GAPDH (compared with control; Fig. 3). The activation status of Akt signaling was subsequently evaluated. CI treatment significantly decreased p-Akt levels and the p-Akt/total Akt ratio in FaDu cells (compared with control; Fig. 3). These findings indicated that CI treatment is associated with reduced EIF2AK3 expression and decreased Akt activation in FaDu cells.

Given the observed alterations in EIF2AK3 expression and Akt activation, the effect of CI treatment on the expression of the anti-apoptotic protein Bcl-2 was subsequently investigated. Western blotting of FaDu cells treated with 1 or 2 mg/ml CI for 24 h revealed a notable concentration-dependent reduction in Bcl-2 protein levels compared with vehicle-treated controls (Fig. 4A). Densitometric analysis further determined the significant progressive reduction in Bcl-2 expression (Fig. 4B), indicating that CI treatment is associated with decreased expression of this anti-apoptotic protein.

To determine whether the observed molecular alterations were accompanied by changes in cell viability, FaDu cells were evaluated using a CCK-8 assay. Cells were exposed to increasing concentrations of CI, and cell viability was assessed after 48 and 72 h of treatment. CI treatment notably reduced FaDu cell viability in a concentration-dependent manner. Compared with the vehicle control, CI significantly reduced cell viability at 1 mg/ml (P<0.05) and at 2 and 5 mg/ml (P<0.01) at both 48 and 72 h. The IC₅₀ values of CI extract were 1.83 mg/ml at 48 h and 1.19 mg/ml at 72 h. The reduction in cell viability was also time dependent, with 72 h of treatment producing a greater decrease than 48 h of treatment relative to the vehicle control, which was defined as 100% viability (Fig. 5). Collectively, these findings demonstrated that CI decreases FaDu cell viability in a concentration- and time-dependent manner and further support its potential cytotoxic activity against HSCC cells.