early apoptosis rose to 14. 32 % from 0. 37 %, and late apoptosis increased to 4. 39 % from 0. 09 %. necrosis also increased by 2. 5 - fold ( 2. 84 % vs. 1. 15 % in the control ), indicating a broad cytotoxic effect on hela cells. these findings reinforce the potential of a. incana dcm fraction as an effective anticancer agent by simultaneously inducing cell cycle arrest and apoptosis. to elucidate the molecular mechanisms behind these effects, we evaluated the expression of apoptotic regulators, including p53, bcl - 2, and bax, through rt - pcr. the intrinsic apoptotic pathway is regulated by the balance between pro - apoptotic bax and anti - apoptotic bcl - 2, where bcl - 2 stabilizes the mitochondrial membrane and prevents cytochrome c release, whereas bax promotes apoptosis by facilitating cytochrome c release [ 21 ]. p53, a critical tumor suppressor, enhances bax expression, thus its stimulation tipping the balance towards apoptosis [ 22 ]. in our study, the a. incana dcm fraction significantly upregulated bax and p53 while downregulating bcl - 2, resulting in an elevated bax / bcl - 2 ratio, a key indicator of apoptosis activation. previous research reported that elevated p53 expression and a higher bax / bcl - 2 ratio correlate with increased tumor sensitivity to anticancer therapies [ 23 ], further supporting the potential of the a. incana dcm fraction as a therapeutic agent. given these findings, it is essential to contextualize the anticancer activity of the a. incana dcm fraction by comparing it to established cervical cancer treatments, such as cisplatin and paclitaxel. cisplatin exerts its cytotoxic effects primarily through dna cross - linking, causing extensive dna damage that leads predominantly to g2 / m cell cycle arrest and subsequent apoptosis induction mainly via p53 - dependent pathways [ 24, 25 ]. despite its efficacy, cisplatin is often limited by severe adverse effects, including nephrotoxicity, neurotoxicity, and drug resistance [ 25 ]. paclitaxel, another cornerstone treatment, acts by stabilizing microtubules, causing prolonged g2 / m mitotic arrest and promoting apoptosis through a p53 - independent pathway involving cyclin b1 / cdc2 activation and bcl - 2 phosphorylation [ 24, 26 ]. however, paclitaxel similarly has clinical limitations, including dose - dependent toxicities such as peripheral neuropathy and myelosuppression [ 27 ]. in contrast, our findings demonstrate that the a. incana dcm fraction distinc tly arrests cells at the g0 / g1 phase and promotes apoptosis via a p53 - dependent pathway. these unique molecular characteristics suggest a complementary role alongside traditional chemotherap eutics, potentially reducing toxicities and overcoming drug resistance, despite its relatively high ic50. the chemical profile of the a. incana dcm fraction, analyzed via gc - ms / fid detection, revealed a high content of phytol ( 65. 74 % ) in unsaponifiable matter, a compound known for its broad - spectrum biological activities, particularly in cancer therapy. phytol, a diterpenoid derived from chlorophyll, has been widely recognized for its anti-inflammatory and anticancer properties, including the modulation of cell cycle and apoptosis pathways [ 28 ]. previous studies have demonstrated its ability to induce apoptosis across various cancer cell lines, including lung, colon, and breast cancers, primarily through the disrup tion of mitochondrial membrane potential and subsequent activation of the intrinsic apoptotic pathway [ 28, 29 ]. our findings, which show significant g0 / g1 cell cycle arrest and apoptosis in hela cells, align with these established mechanisms of action, further validating phytol ’ s role as a potent anticancer agent. in addition to phytol, the saponifiable matter of the dcm fraction contained palmitic acid ( 31. 27 % ) and linolenic acid ( 26. 98 % ) as the major components. both fatty acids are well - documented for their apoptosis - inducing capabilities. palmitic acid, for instance, has been reported to upregulate pro - apoptotic proteins such as bax and p53, while downregulating anti - apoptotic bcl - 2, leading to enhanced apoptosis via caspase activation in colorectal and breast cancer models [ 30 ]. similarly, linolenic acid exerts anticancer effects by modulating the bcl - 2 family proteins, increasing pro - apoptotic bax expression, and inducing endoplasmic reticulum ( er ) stress, which contributes to cancer cell death. linolenic acid has also been shown to inhibit the pi3k / akt signaling pathway and suppress fatty acid synthase ( fasn ), an enzyme overexpressed in many cancers [ 31 ]. our study ’ s findings of increased bax expression and a heightened bax / bcl - 2 ratio in hela cells are consistent with these mechanisms, suggesting that palmitic and linolenic acids likely contribute to the cytotoxic effects observed. despite these promising results, the study possesses limitations typical of in vitro investigations. notably, the biological complexity of tumor microenvironments, such as gene expression variability, signaling heterogeneity, immune interactions, and stromal influences, cannot be adequately mimicked using isolated cell lines like hela. additionally, the relatively high ic50 necessitate s exploration of advanced drug delivery systems or combination therapies. future research should incorporate additional cancer cell lines, normal cell toxicity assessments, patient - derived xenografts ( pdx ), and innovative delivery strategies, such as nanoparticle encapsulation, to enhance efficacy, reduce toxicity, and facilitate clinical translation. these steps are crucial for validating the preclinical promise and advancing the clinical applicability of the alnus incana dcm fraction. conclusion this study demonstrates that the a. incana dcm fraction, rich in bioactive compounds such as phytol, palmitic acid, and linolenic acid, exerts notable cytotoxic effects on various cancer cell lines, particularly hela cells. these effects are mediated through inducing g0 / g1 cell cycle arrest and apoptosis via modulation of bax, bcl - 2, and p53 pathways. although the relatively high ic50 value ( 135. 6 µg / ml ) limits its potential as a standalone therapy, the fraction ’ s selective cytotoxicity toward cancer cells suggests a potentially favorable safety profile. its unique ability to activate apoptotic and cell - cycle arrest pathways supports its promise as an adjunctive therapy, particularly in combination with conventional chemotherap eutic agents, which may improve treatment outcomes by reducing associated toxicities or overcoming resistance. further in - depth mechanistic studies, along with rigorous in vivo evaluations using clinically relevant models such as patient - derived xenografts, are essential to validate these promising findings and fully realize the clinical potential of this natural fraction. introduction antiretroviral therapy ( art ) has been highly effective at suppressing human immunode ficiency virus ( hiv ) replication, thus significantly reducing disease progression and mortality in infected individuals. however, art is not curative and does not meaningfully impact persistent viral reservoirs ( vrs ) of latently infected cells that comprise cd4 + t cells and myeloid cells. 1, 2, 3 indeed, upon antiviral treatment interruption ( ati ), the virus quickly rebounds after several weeks, highlighting how the presence of vrs obviates an hiv cure. 4, 5, 6 therefore, eliminating or controlling vrs remains an unwavering priority for hiv cure research.