the saponifiable fraction presented a balanced composition of saturated ( 48. 69 % ) and unsaturated ( 51. 29 % ) fatty acids, with palmitic acid, linolenic acid, and linoleic acid as the predominant compounds. conclusion while the dcm fraction ’ s relatively high ic50 value may limit its utility as a standalone treatment, its ability to induce cell cycle arrest and apoptosis demonstrates its promise as a co - therapeutic agent with conventional anticancer drugs. further research is essential to elucidate its precise mechanisms of action and to evaluate its efficacy in combination therapies, potentially advancing its role in cervical cancer treatment. methods cell cycle analysis to assess the impact of a. incana dcm fraction on cell cycle progression, propidium io dide ( pi ) staining followed by flow cytometry was employed as outlined previously [ 17 ]. pi ( company ) is a fluorescent dye that binds to dna allowing differentiation of cell cycle phases ( g0 / g1, s, g2 / m ) based on their dna content. briefly, hela cells ( 2 × 105 cells / ml ) were cultured overnight and then treated with the dcm fraction at its ic50 concentration ( 135. 6 µg / ml ) for 24 h. after treatment, cells were fixed, stained with a pi / rnase solution, and analyzed using a facscalibur scan flow cytometer ( bd biosciences ) to determine cell cycle distribution. apoptosis analysis using annexin v - fitc / pi assay to determine the mode of cell death induced by a. incana dcm fraction, the annexin v - fitc apoptosis detection kit ( biovision inc., ca, usa, k101 - 25 ) was used following the manufacturer ’ s protocol. this assay detects phosphatidylserine translocation, a hallmark of early apoptosis, by annexin v - fitc staining. pi, a viability dye excluded by healthy and early apoptotic cells with intact membranes, stains the dna of late apoptotic and necrotic cells with compromised membranes, allowing for their identification. briefly, hela cells ( 2 × 105 cells / ml ) were cultured for 24 h and treated with the extract at its ic50 concentration ( 135. 6 µg / ml ) for an additional 48 h. following treatment, cells were stained with annexin v - fitc and pi. apoptosis was quantified by flow cytometry using a bd facscalibur scan system ( bd biosciences ). for ac curate quantification of apoptotic populations, a specific gating strategy was applied. cells were initially gated based on forward scatter ( fsc ) versus side scatter ( ssc ) to exclude debris, followed by gating on fsc - height ( fsc - h ) versus fsc - area ( fsc - a ) to exclude doublets. apoptotic populations were classified by gating on annexin v - fitc versus pi as follows: live cells ( q4 ) were annexin v - / pi -, early apoptotic cells ( q3 ) were annexin v + / pi -, late apoptotic / necrotic cells ( q2 ) were annexin v + / pi +, and dead / necrotic cells ( q1 ) were annexin v - / pi +. the percentage of cells in each quadrant was calculated to assess the extent of apoptosis and necrosis. statistical analysis statistical analysis was performed using spss version 24. 0. differences between the means of two independent groups were analyzed using an independent t - test, with p * * * results * * * effect of a. incana dcm fraction on cell cycle analysis to investigate the effect of the a. incana dcm fraction on cell cycle progression, hela cells were treated with the ic50 concentration ( 135. 6 µg / ml ) of the fraction for 48 h. cell cycle distribution was assessed by flow cytometry using propidium io dide ( pi ) staining, which binds to dna and allows differentiation of cell cycle phases ( g0 / g1, s, g2 / m ) based on dna content. as illustrated in fig. 2, a significant increase ( p a. incana exerts cytostatic effects on hela cells by inducing cell cycle arrest. fig. 2flow cytometric analysis of cell cycle distribution in hela cells treated with the a. incana dcm fraction. hela cells were treated with the ic50 concentration ( 135. 6 µg / ml ) of the a. incana dcm fraction for 48 h, and cell cycle phase distribution was analyzed via flow cytometry. panels: ( a ) control ( untreated ) hela cells, ( b ) hela cells treated with the dcm fraction, and ( c ) a graphical comparison of cell percentages across each cell cycle phase between treated and untreated groups discussion this study investigated the cytotoxic potential of the dcm fraction from the leaves of the medicinal plant a. incana. given the documented cytotoxic activity of the dcm extract from the related subspecies a. rugosa [ 8 ], we hypothesized that a. incana might exhibit similar effects. although previous studies confirmed the cytotoxic properties of its methanol extract [ 10 ], the effects of the dcm fraction remain unexamined. to address this gap, the fraction ’ s cytotoxicity was evaluated against four human cancer cell lines: hepg2, hct116, hela, and mcf - 7, representing diverse cancer types. the a. incana dcm fraction demonstrated ic50 values ranging from 135. 6 to 273. 1 µg / ml, with hela cells ( a cervical cancer model ) showing the highest sensitivity ( ic50 = 135. 6 µg / ml ). significantly, the dcm fraction exhibited a selectivity index ( si ) of 2. 72 against hela cells, indicating preferential cytotoxicity toward cancer cells over normal cells and highlighting its therapeutic potential. although the ic50 values for a. incana were higher than those reported for the dcm extract of the related subspecies a. rugosa [ 8 ], this difference may be attributed to variations in the concentration or composition of active compounds in each extract. despite this, both species showed their most potent cytotoxic effects against hela cells, suggesting a potential shared mechanism targeting cervical cancer cells specifically. additionally, prior studies have reported that the methanol extract of a. incana leaves has a pronounced effect on hela cells ( ic50 of 68. 5 µg / ml ) [ 10 ]. the selective cytotoxicity observed in both a. incana and a. rugosa dcm extracts, alongside the methanol extract ’ s activity, indicates the presence of bioactive compounds within these species that may preferentially disrup t hela cell function. this selective activity underscores the potential of alnus species as promising candidates for developing targeted therapies against gynecological cancers, particularly cervical cancer. given the substantial cytotoxic response observed, further investigation into the active compounds and mechanisms of action of the dcm fraction of a. incana leaves is warranted. to explore the mechanisms underlying this cytotoxicity, we investigated the effects of a. incana dcm fraction on cell cycle progression and apoptosis in hela cells. the results revealed a significant accumulation of cells in the g0 / g1 phase, indicating effective cell cycle arrest. this is a crucial finding science cell cycle arrest at key checkpoints is a well - established therapeutic strategy for inhibiting cancer cell proliferation. the mammalian cell cycle comprises four distinc t phases: g1, s, g2, and m, where rna and protein synthesis occur in g1 and g2, dna replication in s, and chromosome segregation in m [ 18 ]. by halting cells in the g0 / g1 phase, the a. incana dcm fraction appears to block progression to dna synthesis, which could lead to senescence or cell death. several plant - derived anticancer agents, such as taxanes and vinca alkaloids, function by disrup ting key cell cycle regulators [ 19 ]. in addition to inducing cell cycle arrest, the dcm fraction significantly promoted apoptosis, a fundamental mechanism in cancer treatment. apoptosis, or programmed cell death, is triggered by many natural cytotoxic agents, both clinically approved and experimental, and is essential for eliminating cancer cells [ 20 ]. flow cytometric analysis using annexin v and propidium io dide staining revealed a marked increase in apoptotic cell death ( 40. 67 - fold increase, 18. 71 % vs. 0. 46 % in the control ) induced by the a. incana dcm fraction.