The plant Tragopogon dubius was harvested at morning time during June from the elevated mountainous region of the village Lajoora from the Pulwama district, Jammu & Kashmir, India (33°52^′21.13^″ N, 74°53^′34.27^″ E, and an altitude of 1669.0 m above sea level). Mr. Akhtar Malik performed taxonomical identification at the University of Kashmir. A voucher specimen (No.: 2940-(KASH)) was preserved at the Centre of Taxonomy and Biodiversity, University of Kashmir, Hazratbal, Srinagar, India. Fresh leaves and stems were dried at 40°C for 12 h in an oven, then finely powdered and stored in airtight glass containers for subsequent analysis.

Dimethyl sulfoxide (DMSO, ≥99.9%), ethidium bromide (EtBr, ≥95%), sodium carbonate (99.5%), Folin–Ciocalteu reagent (98%), aluminium chloride (99.5%), nicotine amide adenine dinucleotide (NADH, 98%), hydrogen peroxide (H₂O₂, 97%), sodium hydroxide (97%), nitro blue tetrazolium (NBT, 95%), phenazine methosulfate (PMS, 95%), low melting point agarose (LMPA, 98%), nicotine amide adenine dinucleotide (NADH, 98%), normal melting point agarose (NMPA, 98%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%), were purchased from HiMedia laboratories Pvt. Ltd., Mumbai, India. Gallic acid (99%), HPLC-grade methanol (99%), DPPH (98%), and rutin (99%) were procured from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 (99%) and DMEM (99%) media were obtained from Merck Sigma Aldrich. All bioassays were conducted using analytical grade reagents.

One hundred grams of the finely powdered leaves and stems of Tragopogon dubius were each placed into separate conical flasks and extracted with 500 mL of double-distilled autoclaved water at room temperature for 24 h under continuous stirring using a magnetic stirrer. The resulting solutions were filtered through Whatman filter paper, producing black-colored filtrates. To ensure that no residue passed through, the filtrates were centrifuged at 3000× g. The clear solutions were then concentrated to near dryness using a rotary evaporator. The semi-solid extracts were air-dried at 40°C for 5 days to eliminate residual moisture, yielding 1.4 g of AQ-TrDL (leaf extract) and 2.5 g of AQ-TrDS (stem extract). The extracts were stored at 4°C for further analysis. The extraction method followed that of [24], with slight modifications.

The Folin-Ciocalteu method for total phenolic content (TPC) determination and aluminium chloride method for total flavonoid content (TFC) were employed for the AQ-TrDL and AQ-TrDS extracts, following the protocol adopted by Chandni et al. with slight modifications [25]. Gallic acid and rutin served as the standard compounds, with TPC expressed as milligrams of gallic acid equivalents per gram of dry extract weight (mg GAE/g DW) and TFC as milligrams of rutin equivalents per gram of dry extract weight (mg RE/g DW). The chemicals and reagents required encompass folin-Ciocalteu reagent, sodium carbonate, aluminium chloride, sodium nitrite, sodium hydroxide, double-distilled water, and methanol.

The cytotoxic efficacy of crude aqueous leaf and stem extracts from Tragopogon dubius were assessed against several human cancer cell lines, including MCF-7 (breast), HCT-116 (colon), HeLa (cervical), and A549 (lung), as well as a normal mouse fibroblast cell line (L-929), utilizing the MTT assay as per standard protocols [30]. All cell lines were procured from NCCS Pune, India, and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% Fetal Bovine Serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37°C in a 5% CO₂ humidified incubator under controlled conditions. Cell line selection was based on initial screening for extract sensitivity, specificity, and selective cytotoxicity, which supported the in vivo extrapolation of the in vitro results. Cancer cells were seeded at a density of 8 × 10³ cells per well in 96-well plates and allowed to adhere for 24 h. Cells were subsequently exposed to varying concentrations (15.125–500 μg/mL) of the AQ-TrDL and AQ-TrDS extracts for an additional 24 h. Following treatment, cells were incubated with 20 μL of MTT solution (5 mg/mL) for 3 h, facilitating the formation of formazan crystals. These were then dissolved by adding 100 μL of DMSO, after which absorbance was recorded at 570 nm. Each concentration was tested in triplicate for each cell line studied, to ensure reliability, reproducibility, and for the calculation of standard deviation and statistical significance. The GI₅₀ values, representing the concentration of extracts required to inhibit 50% cell growth, were calculated using the following formula: % Growth Inhibition=(A0−A1)A0×100 where A₀ is the absorbance of untreated control cells, and A₁ is the absorbance of treated cells at various extract concentrations.

Based on cytotoxicity results, the AQ-TrDS extract was found to exhibit superior anticancer activity against MCF-7, HCT-116, HeLa, and A549 cell lines. The apoptotic effects of AQ-TrDS on these cancer cells were further investigated using fluorescence microscopy, with a focus on mitochondrial and nuclear alterations.

DAPI and Rhodamine Staining

Cancer cells were seeded into 24-well plates at an optimal density of approximately 1 × 10⁵ cells/well and incubated overnight to facilitate adhesion and proliferation, as previously described by [25]. Each well was then exposed to a GI50 concentration of the AQ-TrDS extract for 24 h. After the completion of the treatment period, the media containing unattached dead cells and AQ-TrDS was removed. The treatment period, the media containing unattached dead cells and AQ-TrDS was removed, and each well was washed with 1× PBS (Phosphate-Buffered Saline). After washing, 0.490 μL of 1× PBS was added again to each well, where 10 μL of DAPI (Stock solution, 1 μg/mL) and 10 μL of Rhodamine 123 (stock solution, 1 μg/mL) were mixed. The mixture was then allowed to incubate in the dark for 30 min inside a CO₂ incubator. After incubation with respective dyes, the excess dyes were removed by washing gently twice with 1× PBS, followed by visualizing through a fluorescence microscope (Nikon Eclipse T2, Japan) to observe nuclear alterations and loss of mitochondrial membrane potential.

The alkaline comet assay, based on the protocol by [31] was used to evaluate the genotoxic potential of AQ-TrDL and AQ-TrDS extracts. Briefly, 1 × 10⁴ cells were exposed to various concentrations (40, 80, 160, and 320 μg/mL) of the extracts for 30 min at 37°C in the dark. H₂O₂ (5 μg/mL) was employed as a positive control to induce oxidative DNA damage. To assess the genoprotective capacity, extracts were co-incubated with H₂O₂. Following treatment, cells were embedded in 0.5% low-melting-point agarose (LMPA) on slides pre-coated with 1% normal-melting-point agarose, solidified at 4°C, and later immersed in lysis buffer (pH 10) for 4 h at 4°C. DNA migration was induced by electrophoresis at 25 V and 300 mA for 30 min. After neutralization and staining with ethidium bromide, comets were visualized under a Nikon Eclipse Ts2 fluorescence microscope (Japan). DNA damage was quantified using Comet Assay Software Project (Casp lab).

Amino acid composition was analysed utilizing an Agilent 1260 series High-Performance Liquid Chromatography (HPLC) system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector, as per the standardized Agilent protocol. The system incorporated an Agilent Advance Bio AAA column (4.6 × 100 mm) housed within a thermostatically controlled column compartment maintained at 40°C. Sample preparation was conducted through a standardized protocol involving 24 h hydrolysis at 110°C using 6 M hydrochloric acid (HCl) to yield protein hydrolysates. The amino acids were separated and quantified via pre-column derivatization, utilizing FMOC (fluorenyl methoxycarbonyl) chloride for primary amino acids and OPA (o-phthaldialdehyde) for proline. Calibration was performed using a standard amino acid mixture (0.5 μM/mL for each amino acid). The mobile phase consisted of 40 mM sodium phosphate buffer (pH 7.8) as phase A, and a mixture of acetonitrile, methanol, and water (45:45:10 v/v) as phase B. A 2.5 μL sample injection was employed, with separation achieved at a flow rate of 2 mL/min. The gradient program began with a 0% phase B for 1.9 min, followed by an increase to 53% phase B over 16.3 min. A washing step at 100% phase B and re-equilibration at 0% phase B concluded the run, with a total analysis time of 26 min. Each sample was analysed in triplicate [32–34].

Mineral content in the leaves and stems of T. dubius was determined using the acid digestion method. One gram of dried, ground seed sample was placed in a 250 mL conical flask, followed by the addition of 20–30 mL of a tri-acid mixture (HNO₃:H₂SO₄:HClO₄ = 10:1:4). The contents were gently stirred to ensure thorough moistening of the sample, then heated on an electric hot plate at 180–200°C until white fumes appeared, and the solution became colorless. Upon partial drying or yellowing of the solution, an additional 5 mL of the tri-acid mixture was added. Once digestion was completed, the flask was removed from the heat and allowed to cool. Subsequently, 20–30 mL of distilled water was added, and the contents were thoroughly mixed. The solution was then filtered using Whatman No. 1 filter paper into a 100 mL volumetric flask, with repeated washing of the conical flask to ensure complete transfer of minerals [35].

Elemental Analysis via Microwave Plasma Atomic Emission Spectroscopy (MP-AES)

Elemental analysis was conducted using an Agilent 4210 Microwave Plasma Atomic Emission Spectroscopy (MP-AES) system (Agilent Technology Company), equipped with a standard MP-AES torch, concentric nebulizer, and glass cyclonic spray chamber. The nebulizer flow rate ranged from 0.45 to 0.95 L/min, with an uptake time of 70 s at 80 rpm. Measurements were taken in triplicate, with a stabilization time of 5 s.

The aqueous extracts of T. dubius AQ-TrDL and AQ-TrDS were analysed using gas chromatography-high resolution mass spectrometry (GC-HRMS). An Agilent 7890 Gas Chromatograph, coupled with a JEOL AccuTOF GCV JMS-T100GCV Mass Spectrometer, was utilized. Helium served as the carrier gas at a flow rate of 1.0 mL/min. Separation was achieved using an HP-5 capillary column (30 m μm film thickness). Samples diluted in acetone at a 1:100 v/v ratio, were injected with a split ratio of 1:10 for 1 min, maintaining the injector temperature at 250°C. The column temperature was initially set at 60°C for 2 min, followed by a rise to 250°C, then increased to 280°C at a rate of 30°C/min and held for 10 min. Mass spectra were recorded within a mass range of 45–650 amu at energy of 70 eV. Identification of the constituents was based on the obtained mass of peaks with library matches.

Fourier-transform infrared (FTIR) spectroscopy was employed to analyse the AQ-TrDL and AQ-TrDS aqueous extracts. Spectra were recorded in absorbance mode using a Shimadzu FTIR spectrometer (Model IR Tracer-100), covering a frequency range of 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹. FTIR analysis provided detailed insights into the functional groups present in the aqueous extracts by analysing distinct absorption bands, facilitating a comprehensive characterization of their chemical composition [36].

Statistical Analysis

Experiments were carried out in triplicate, and for statistical analysis, data were presented as the Mean ± standard deviation. One-way Analysis of Variance (ANOVA) with a significance level set at p ≤ 0.05 was used to calculate significant differences among the data variables. Tukey’s HSD (Honestly Significant Difference) test, conducted through post hoc comparisons, was used to assess and compare the means. The standard curve parameters were used to calculate GI₅₀ and IC₅₀ values using Excel software (2019) by comparing data variables in triplicate.

In the present investigation, the phenolic and flavonoid concentrations in the aqueous extracts AQ-TrDL and AQ-TrDS of T. dubius were analysed and are presented in Table 1. The AQ-TrDS extract exhibited a significantly higher phenolic content of 225.63 ± 2.23 mg GAE/g extract, compared to the AQ-TrDL extract, which recorded 74.25 ± 1.98 mg GAE/g extract. Similarly, the total flavonoid content was more abundant in AQ-TrDS, with a conentration of 121.25 ± 3.01 mg RE/g extract, while AQ-TrDL showed a lower flavonoid content of 61.62 ± 1.10 mg RE/g extract. The significant levels of phenolic and flavonoid content underscore the extract’s promise as a natural source of bioactive compounds for pharmaceutical or nutraceutical applications.

In this study, the antioxidant potential of the AQ-TrDL and AQ-TrDS aqueous extracts of T. dubius were evaluated using DPPH, superoxide, FRAP, and ABTS radical scavenging assays. The IC₅₀ values and inhibition percentages for these assays are summarized in Table 2. The results revealed concentration-dependent antioxidant potential at concentrations ranging from 25 to 800 μg/mL, as illustrated in Fig. 1. The AQ-TrDS extract consistently exhibited the and most potent antioxidant activity across all assays, with IC₅₀ values of 73.58 μg/mL (DPPH), 43.47 μg/mL (superoxide), 50.56 μg/mL (FRAP), and 40.31 μg/mL (ABTS), and corresponding inhibition percentages of 87.53%, 92.05%, 86.65%, and 92.61%, respectively. In contrast, the AQ-TrDL extract an IC₅₀ value of 173.19 μg/mL. The reference standard, rutin, demonstrated IC₅₀ values of 28.35, 32.14, 36.96, and 29.54 μg/mL in the DPPH, superoxide, FRAP, and ABTS assays, respectively. The potent antioxidant activity observed in AQ-TrDS supports the extract’s potential as a natural therapeutic source against oxidative stress-related conditions.

The anticancer efficacy of AQ-TrDL and AQ-TrDS extracts was evaluated against MCF-7 (breast cancer), HCT-116 (colon cancer), HeLa (cervical cancer), and A-549 (lung cancer) cell lines, as well as the normal fibroblast L-929 cell line, using the MTT assay in vitro. Among the tested extracts, AQ-TrDS demonstrated the highest cytotoxicity, inhibiting more than 50% of cell proliferation in MCF-7, HCT-116, HeLa, and A-549 cell lines at concentrations of 56.62, 90.37, 77.73, and 98.32 μg/mL, respectively. The inhibitory effects of AQ-TrDS on cancer cell viability were observed to be concentration-dependent (Fig. 2), with maximum growth inhibition rates of 89.81%, 81.35%, 81.33%, and 87.78% at the highest tested concentration of 500 μg/mL in the respective cancer cell lines. In contrast, AQ-TrDL exhibited comparatively lower cytotoxic activity, with a GI₅₀ value of up to 166.78 μg/mL in the A-549 cell line. The GI₅₀ values for all tested cell lines are presented in Table 3. Significant variations in cytotoxicity were observed between AQ-TrDS and AQ-TrDL, suggesting differential efficacy against the selected cancer cell lines. Notably, both extracts exhibited minimal toxicity towards the normal fibroblast L-929 cell line, with GI₅₀ values of 522.31 μg/mL and 434.25 μg/mL for AQ-TrDL and AQ-TrDS, respectively. The potent antiproliferative and cytotoxic effects of the extract depict its valuable bioactive constituents and underscore its potential for developing plant-based natural chemotherapeutic agents.

Given that the aqueous extract of Tragopogon dubius (AQ-TrDS) demonstrated superior antiproliferative and cytotoxic effects on the aforementioned cancer cell lines, fluorescence microscopy was employed to further assess apoptotic induction. DAPI staining was utilized to visualize alterations in nuclear morphology as depicted in Fshow. In contrast, Rhodamine 123 staining was applied to monitor mitochondrial membrane potential at given in Fig. 3. In contrast, Rhodamine 123 staining was applied to monitor mitochondrial membrane potential as shown in Fig. 4 at the GI₅₀ concentration of the extract. The findings revealed that treatment with AQ-TrDS extract induced significant nuclear alterations, including membrane blebbing, shrinkage, and fragmentation—hallmarks of apoptotic processes within cancer cells. Furthermore, the analysis of mitochondrial membrane potential showed a marked disruption following exposure to the extract, evidenced by a substantial reduction in fluorescence intensity compared to untreated control cells. These morphological aberrations and the loss of mitochondrial membrane potential corroborate the hypothesis that AQ-TrDS extract contains bioactive phytochemicals capable of inducing apoptosis. Thus, these results advocate for the further isolation and characterization of Tragopogon dubius phytochemicals for potential therapeutic applications in cancer treatment.

The genotoxicity and genoprotective potential of AQ-TrDL and AQ-TrDS aqueous extracts were assessed using the alkaline comet assay on human blood lymphocytes at concentrations of 40, 80, 160, and 320 μg/mL. This assay aimed to evaluate the extent of DNA damage. Our results revealed that none of the tested concentrations of AQ-TrDL or AQ-TrDS induced significant DNA fragmentation or comet formation in human lymphocytes, indicating the absence of genotoxic properties in these extracts. The morphology of the treated lymphocytes remained essentially spherical, consistent with the negative control group, thereby substantiating the lack of notable genotoxic effects (Fig. 5). Hydrogen peroxide (“H₂O₂”) was used as the positive control, effectively inducing DNA damage in blood lymphocytes, as demonstrated in Fig. 5Ai, Bi. The treated lymphocytes were analysed for tail length (TL), olive tail movement (OTM), and percentage tail DNA content (% TDNA). No statistically significant alterations were observed in these parameters when compared to the control group, as presented in Tables 4 and 5. However, both AQ-TrDL and AQ-TrDS extracts exhibited a substantial protective effect against “H₂O₂”—induced DNA damage in lymphocytes, with AQ-TrDS demonstrating a more pronounced effect even at the lowest tested concentration (40 μg/mL). This enhanced protective activity may be attributed to the presence of bioactive phytochemicals in the extract. The extent of DNA damage induced by “H₂O₂” initially recorded at 69.85% was progressively reduced to 40.625%, 29.144%, 19.647%, and 8.668% by AQ-TrDS at concentrations of 40, 80, 160, and 320 μg/mL, respectively. The non-genotoxic and strong genoprotective profile of AQ-TrDS supports its relevance for developing safe pharmaceutical and nutraceutical formulations, but much in-depth toxicological investigations through animal models are necessary to validate the extract’s biosafety potential.

The amino acid profile of T. dubius leaves and stems was meticulously analysed, revealing the presence of several key amino acids: aspartic acid, glutamic acid, glycine, threonine, arginine, alanine, tyrosine, cystine, valine, tryptophan, phenylalanine, and leucine. These amino acids were precisely identified and quantified using High-Performance Liquid Chromatography (HPLC), with concentrations reported as milligrams per gram of protein. Chromatograms are shown in micrograms per gram of protein. Chromatograms are shown in micrograms per gram of protein. The chromatograms are shown in Fig. 6. The detailed results, including peak number, area, retention time, and amino acid concentration for both leaf and stem samples, are provided in Tables 6 and 7. In total, 10 amino acids were identified in the leaf samples, while 14 were detected in the stem samples of T. dubius.

The elemental composition of Tragopogon dubius leaves and stems was analysed using Microwave Plasma-Atomic Emission Spectroscopy (MPAES), with the results summarized in Table 8. The analysis reveals the presence of several major elements, including sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), bismuth (Bi), aluminium (Al), and iron (Fe). Trace elements such as manganese (Mn), copper (Cu), zinc (Zn), lithium (Li), and nickel (Ni) were also detected. Furthermore, the presence of potentially toxic heavy metals, such as lead (Pb) and chromium (Cr), was noted; however, their concentrations were minimal and remained within the permissible limits established by the FAO/WHO guidelines. Notably, the elemental concentrations varied between the leaf and stem tissues [37]. The leaves exhibited elevated levels of iron, calcium, potassium, magnesium, aluminium, and sodium. In contrast, the stems showed high concentrations of only calcium, potassium, and magnesium. The detection of vital elements enhances the dietary value of Tragopogon dubius and can be added in developing plant-based mineral-enriched supplements and herbal formulations.

The GC-HRMS analysis revealed a diverse array of phytochemicals in the aqueous extracts of Tragopogon dubius AQ-TrDL and AQ-TrDS). Distinct chromatographic peaks in the aqueous extracts corresponded to various phytoconstituents. Specifically, the AQ-TrDL extract was found to contain 18 phytochemical constituents, while the AQ-TrDS extract harbored 23 distinct constituents; their respective chromatograms are presented in Fig. 7. Prominent phytochemicals identified in the AQ-TrDL extract include quinic acid (11%), 1,3-propanediol, 2-(hydroxymethyl)-2-nitro (12.52%), and 9-Octadecenoic acid (Z)-, methyl ester (10.3%). Conversely, the AQ-TrDS extract was dominated by (-)-Globulol (14%), Tridecane (11.24%), and n-Hexadecenoic acid (13.52%). Collectively, these extracts encompass a broad spectrum of chemical classes, including triterpenoids, fatty acids, carboxylic acids, diterpenes, alcohols, and esters. Comprehensive details regarding the active principles, including their retention times (RT), molecular formulas, molecular weights, and concentrations (expressed as peak area %), are provided in Tables 9 and 10. The rich diversity of phytochemicals validate that Tragopogon dubius leaves and stems as valuable sources for pharmaceutical and nutraceutical applications.

The infrared (IR) absorption spectra of the aqueous extracts from the leaves and stems of T. dubius are depicted in Fig. 8. The major peaks observed in the FTIR spectra correspond to various functional groups, including alcohols, alkanes, alkenes, alkynes, aldehydes, amines, carboxylic acids, esters, sulfoxides, hydroxyl groups, and alkyl halides. Table 11 summarizes the functional groups linked to these peaks in the FTIR spectra of the leaf and stem extracts, which align with the phytoconstituents identified through GC-HRMS analysis.