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INTRODUCTION
Cancer denotes the most difficult universal health challenges of the 21st century place as the 2nd leading cause of death worldwide with an estimated 19.3 million new cases and 10 million cancer associated losses reported in 2020 (Sung et al., 2021). Cervical cancer is a malignancy of hematopoietic tissue characterized by uncontrolled proliferation of immature blood cell precursors constitutes a significant proportion of blood related cancers with considerable morbidity and mortality across all demographic groups (Choi et al., 2024). Conventional therapeutic modalities including chemotherapy, radiotherapy and surgical while clinically active to variable degrees are regularly associated with severe systemic toxicity, multi-drug resistance and adverse side effect profiles that consider ably reduce quality of life for patient. (Magno et al., 2024). So, the imperative to develop fresh, selectively cytotoxic and biocompatible anticancer agents has intensified substantially within the oncologic research community (Basak et al., 2021). In biomedical applications NPs have been generally discovered for drug delivery, diagnostics, imaging, and therapeutic purposes due to their ability to penetrate biological barriers, improve drug solubility, and enable controlled and targeted release (Jaganmoorthy et al., 2026). The size, shape and surface charge of nanoparticles can be personalized to optimize cellular uptake and therapeutic efficacy while their surfaces can be functionalized with biomolecules to improve specificity to cancer cells and decrease damage to normal tissues (Anandachockalingam et al., 2024). Nanotechnology has developed as a transformative discipline within biomedical research, offering extraordinary opportunities for targeted drug delivery system, diagnostic imaging, and direct therapeutic intervention at the cellular and molecular level (Ishaque et al., 2025). Green development of nanoparticles using herbal extracts has earned high interest as an environmentally friendly, profitable and maintainable another to conservative chemical and physical methods (Lintao and Medina et al., 2021). Plant based synthesis removes the use of toxic reagents and provides natural dropping and stabilizing agents, enhancing the biocompatibility of the nanoparticles (Baskar et al., 2025). Among the varied of nanomaterials below examination metal oxide nanoparticles have gaining attention significant scientific attention to their distinctive physicochemical properties (Khalil et al., 2017). Iron Nanoparticles (FeNPs) in particular have been widely studied in oncologic backgrounds due to their magnetic characteristic demonstrated biocompatibility and volume to produce Reactive Oxygen Species (ROS) within malignant cells through Fenton type reactions thereby inducing oxidative stress mediated cytotoxicity (Limo et al., 2018). The plant based of nanoparticles applying phytochemical extracts has produced substantial momentum as an ecologically sustainable and economically viable another to conventional chemical and physical fabrication methodologies (Osman et al., 2024). Phytoderived reducing agents comprising a complex matrix of polyphenols, flavonoids, terpenoids, alkaloids and organic acids facilitate the decrease of metallic ions and simultaneously function as capping and stabilizing agents thereby obviating the requirement for hazardous chemical reducing agent and minimizing toxic byproduct generation (Singh et al., 2023). The nanoparticles developed by the help of photosynthesis moreover exhibit synergistic biological activities attributable to the retention of bioactive phytochemical ligands on surface of the particles. Schinus molle L., commonly selected as the Peruvian peppertree or pink pepper is a xerophytic aromatic tree distributed across tropical and subtropical regions of South America subsequently naturalized across Africa, Asia and the Mediterranean basin (El-Nashar et al., 2022). The species has been extensively employed in indigenous and traditional medical systems for the treatment of antimicrobial infections, inflammatory conditions, dental caries, respiratory ailments and skin disorders (Fonseca and Huerta-Reyes et al., 2025). Phytochemical study has documented an extraordinarily diverse secondary metabolite profile encompassing essential oil constituents pentacyclic triterpenoids, flavonoids, tannins, phenolic acids and alkaloids many of which possess demonstrated antioxidant, antimicrobial, anti-inflammatory and antiproliferative activities (Radice et al., 2026). The rich polyphenolic and flavonoid content of S. molle renders its water extract particularly suitable as a bio decreasing and capping agent for nanoparticle synthesis (Acharya et al., 2025). The current investigation was designed to investigate the anticancer potential of FeNPs biosynthesized from S. molle leaf aqueous extract against HeLa cells. Specifically, the objectives encompassed (i) Physicochemical characterization of synthesized FeNPs through FTIR spectroscopy to identify functional groups mediating nanoparticle stabilization (ii) Quantitative assessment of cytotoxic activity through the MTT cell viability assay across a defined concentration gradient (iii) Qualitative visualization of cellular morphological alterations and apoptosis induction employing Acridine Orange/Ethidium Bromide dual-fluorescence staining methodology and (iv) Evaluation of the morphological integrity of HeLa cells at the IC₅₀ concentration through phase-contrast microscopy.
MATERIALS AND METHODS
Plant Material Collection and Extract Preparation
S. molle leaves were collected and washed with tap water to remove all unwanted residues and material. After washing the leaves were spread in a shaded place and allowed to dry naturally at room temperature until they become completely dry. After drying the material was crushed manually and powdered using a clean grinder to obtain a fine texture. 10 g of the plant powdered material was placed into a beaker filled with distilled water. The mixture was heated slowly at a mild temperature to allow the bioactive compound to leach into the solvent. After heating the extract was left undisturbed to cool naturally. The cooled mixture was filtered through Whatman filter paper to remove solid residues. Clear extract was collected and kept at 4ºC for further process in the development of nanoparticles (Sarwar et al., 2025).
Biosynthesis of Iron Nanoparticles
Iron nanoparticles were developed through 0.1 molarity water solution of ferric chloride hexahydrate (FeCl₃·6H₂O) was freshly prepared with distilled water. The mixture was accrued by adding 10 mL of S. molle leaf extract add dropwise 90 mL of the FeCl₃ solution under continuous stirring at 60ºC. The preparation of FeNPs was showed by a changing of colour from pale yellow to dark brown black within half an hour of reaction beginning, denoting the reduction of Fe³⁺ ions and nucleation of iron nanoparticles. The combination was kept below stirring for 2 hr at 60ºC for complete reaction. The suspension was transfer to centrifugation at 10,000 rpm for 20 min at 4ºC and the pellet was washed three times with distilled water followed by ethanol to remove residual phytochemicals and unreacted precursors. The nanoparticle which is purified and the pellet was dried in a hot-air oven at 60ºC for 12 hr and stored in airtight container under room temperature for further studies (Elkhateeb et al., 2024).
Fourier Transform Infrared Spectroscopy (FTIR)
Fourier-transform infrared spectroscopy (Nicolet 5700, Thermo Scientific) was used to clarify the of Schinus molle green synthesized Iron Nanoparticles. The KBr pellet method was used to develop the samples, which were then analysed over 500-4000 cm-1. The observed vibration bands confirmed that the nanoparticles were synthesised successfully and that bio-based compounds were present on their surface (Viswanathan et al., 2025).
Surface Morphology using SEM
S. molle developed Iron Nanoparticles (FeNPs) were detected using SEM (JEOL JSM5600LV, Japan). The Nanoparticles was spread on polycarbonate sheets and dried using CO2 critical point drying method to eliminate the remaining moisture. The sample were covered with tinny layer of gold to increase conductivity at before taking picture. The SEM images are possible to get the overall shape, size range and surface morphology of the particles giving a clear idea about their overall formation (Sankar et al., 2026).
Drug Release Study
For Drug release study nanoparticle formulation was dissolved in 10 mL of phosphate buffered saline. The samples were kept incubation at 37ºC under continuous shaking at 100 rpm. At fixed time intervals of 1, 3, 5 and 24 hr, supernatant which is 1 mL was withdrawn and centrifuged at 9,000 rpm for 5 min (Thompson et al., 2024; Iani et al., 2023). An equivalent volume of new buffer was added back to maintain the total volume. Calculation of cumulative percentage of extract released from the nanoparticles was using the following equation:
Cell Culture Maintenance
HeLa cell line was obtained from the National Centre for Cell Science (NCCS), Pune, India, and maintained under standard culture conditions in Dulbecco's Modified Eagle's Medium (HiMedia, India) added with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (antibiotic-antimycotic solution. Cell cultures were kept in a humidified CO₂ incubator at 37ºC with 5% CO₂ atmosphere. Sub-cultivation was performed every 48-72 hr upon attainment of 70-80% confluency utilizing 0.25% trypsin-EDTA solution. All experimental measures were conducted utilizing cells growth phase (passages 5-15) and all manipulations were performed under aseptic conditions within a biological safety cabinet (Gati et al., 2025).
MTT Cell Viability Assay
Nanoparticle induced cytotoxicity was quantified by MTT assay. Cells seeded (1×10⁴/well) in 96-well plates were visible to graded nanoparticle concentrations for 2 days. Post incubation with MTT (0.5 μg/mL, PBS) formazan crystals were solubilized and absorbance was observed at 570 nm. Cell viability percentages were derived accordingly (Paladini et al., 2025).
Morphological Evaluation of HeLa Cells by Phase-Contrast Microscopy
Phase contrast microscopic test of HeLa cell morphology was assumed to assess treatment induced cytological alterations. HeLa cells were sowed in 6-well culture plates at a density of 3×104 cells per well in 2 mL of whole DMEM and incubated for 24 hr to establish adherent monolayers (He et al., 2022).
Acridine Orange/Ethidium Bromide (AO/EtBr) Dual-Fluorescence Staining
HeLa cells were cultured in 6-well plates, treated with FeNPs at the IC₅₀ concentration for 24 hr and then harvested by trypsinization. Cell suspensions (1×104 cells/mL) were prepared in PBS and stained with a freshly prepared AO/EtBr dual dye solution (1:1 v/v combination of 100 μg/mL AO and 100 μg/mL EtBr in PBS) at a ratio of 9:1 (cell suspension: staining solution). Stained cell suspensions (10 μL) were immediately mounted on clean microscopy slides under coverslips and examined under a fluorescence microscope (Alassiri et al., 2025).
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 14.2 and Origin Pro 2025b. Data are presented as mean±Standard Deviation (SD) of at least three independent experiments. Differences among multiple groups were evaluated using One-Way Analysis of Variance (ANOVA) followed by Tukey's post hoc test.
RESULTS
FTIR Spectroscopic Characterization of Biosynthesized FeNPs
Fourier Transform Infrared Spectroscopic analysis of FeNPs was completed to elucidate the nature of functional groups connected with the surface of green produced FeNPs and to classify the phytochemical responsible for nanoparticle decrease, capping and steadying. The FTIR spectrum of S. molle synthesized FeNPs, Figure 1 shows some prominent absorption bands distributed across the midinfrared spectral region (4000-500 cm⁻¹) each corresponding to specific molecular vibrations of surface bound green functional groups. Table 1showing characteristic absorption bands at 3378 cm⁻¹ (O-H stretching), 1634 cm⁻¹ (C=O stretching) and 1087 cm⁻¹ (C-O stretching). Revealed in Figure 2A and Table 1 of surface bound phytochemical capping agents.
| Wavenumber (cm⁻¹) | Vibrational Assignment | Possible Phytochemical Origin |
|---|---|---|
| 3378 | O-H stretching (broad) | Polyphenols, Flavonoids, Adsorbed Water |
| 1634 | C=O stretching / Amide I | Ketones, Carboxylic Acids, Proteins |
| 1087 | C-O stretching | Polysaccharides, Terpenoids, Esters |
SEM Image of Iron Nanoparticles
SEM analysis image revealed that Schinus molle-Synthesized Iron Nanoparticles highly, irregularly clustered morphology of the nanoparticles. The surface presents rough with densely packed granule like formation. The particle size distribution in the image 124 nm to 159 nm result are shown in Figure 2B. Confirming the formation of nanoparticles within the nanoscale range. The particles show a tendency to clustered which may be due to high surface energy and possible interaction between functional groups. Complete the nanoparticle morphology suggests successful nanoparticle formation with a non-uniform, grouped structure which can be beneficial for applications requiring high surface area for drug delivery and catalytic activity.
In vitro Drug release and Cytotoxicity Assessment MTT Assay
In vitro cumulative release profile of Schinus molle-Synthesized Iron Nanoparticles shows a quick initial release within 5 hr and constant release up to 24 hr approaching nearly 100% cumulative release. The drug release from Schinus molle-Synthesized Iron Nanoparticles was tested under controlled conditions and the result are shown in Figure 3A. At starting its was a slow drug release. Nearly 50% of drug was released at 300 min it was a sustainable release. which shows the diffusion of drug molecules from the nanoparticles. So, after 1400 min it shows 70% of drastic release of drug form the nanoparticle.
The cytotoxic possible of bio synthesised FeNPs against HeLa cells was assessed over a different concentration range of 0-100 μg/mL following 24 hr of treatment. The MTT assay results are shown in Figures 3B-3D established pronounced concentration dependent reduction in HeLa cell viability with statistically significant differences observed between all treatment groups relative to the untreated control (p<0.001 for all comparisons by ANOVA with Tukey's post hoc test). Untreated control cells maintained more viability of 97.8±2.3% consistent with normal proliferative activity. Treatment with FeNPs at 5 μg/mL resulted in a marked reduction in cell viability to 49.7±3.1% representing an approximate 49% low relative to untreated controls. Progressive growth in FeNPs different concentration to 10, 25, 50 and 100 μg/mL developed corresponding viabilities of 39.2±2.8%, 31.6±1.9%, 25.3±2.2% and 23.1±1.8% respectively demonstrating a consistent inverse dose-response relationship. The IC₅₀ value defined as the concentration of FeNPs inhibiting 50% of cellular metabolic activity relative to the untreated control was determined to be approximately 5 μg/mL representative potent anticancer activity at low concentrations. The initial rejected in cell viability different ranging between 0 and 5 μg/mL followed by a more gradual at highest concentrations suggests a biphasic cytotoxic mechanism potentially building both direct cellular interactions and indirect ROS mediated pathways.
Cell Morphology
Phase-contrast microscopic study of HeLa cell monolayers providing qualitative assessment of FeNP induced morphological alterations at the IC₅₀ concentration. Untreated control cells (Panel A) showed characteristic HeLa cell morphology showing an adherent, spindle-shaped and polygonal cellular architecture with clearly defined cytoplasmic boundaries, prominent nuclei and extensive intercellular contact consistent with actively proliferating epithelial type cells in confluent monolayer culture. The cells established uniform distribution across the surface of the culture with minute detachment or cellular debris observable in the culture medium. In contrast, HeLa cells screening to FeNPs at the IC₅₀ concentration for 24 hr (Panel B) exhibited apparent morphological modifications indicative of cytotoxic stress. Treated cells were demonstrated partial loss of the characteristic spindle shaped morphology with cells appearing more rounded and exhibiting reduced intercellular contact and adhesion. The overall cell density in FeNPs treated wells seemed diminished relative to untreated controls, constant with inhibition of cell proliferation and induction of cell death. These morphological observations support the quantitative MTT assay data proving the cytotoxic activity of green based FeNPs against HeLa cells.
Acridine Orange/Ethidium Bromide (AO/EtBr) Fluorescence Staining Analysis
AO/EtBr double fluorescence staining was lively to illustrate the mode of cell death encouraged by green based FeNPs in HeLa cells and to distinguish viable apoptotic and necrotic cellular populations based on differential membrane integrity and nuclear morphology. Typical fluorescence photomicrographs of AO/EtBr-stained HeLa cells are shown with Panel A depicting AO channel (Green Fluorescence FITC Filter) and Panel B shows the united AO/EtBr overlay (Orange/Red Fluorescence, Texas Red filter) in FeNPs treated cells. Fluorescence microscopic examination revealed distinct differential staining patterns revealing of apoptotic cell death in FeNPs treated HeLa cells. Under AO channel imaging (Panel A, green fluorescence), cells displayed heterogeneous fluorescence intensity with indication of nuclear condensation and irregular chromatin distribution, characterized by bright green fluorescent foci within nuclei indicative of chromatin compaction in early apoptotic cells. The overlay fluorescence micrograph (Panel B) demonstrated the occurrence of cells exhibiting orange-to-red nuclear fluorescence with condensed, fragmented, or crescent-shaped nuclear morphology, characteristic morphological hallmarks of late apoptosis attributed to EtBr intercalation into DNA of cells with compromised membrane integrity. Specifically the following cellular categories were identified (i) Viable cells displaying uniform bright green fluorescence (AO-positive, EtBr-negative) with normal nuclear morphology (ii) Early apoptotic cells exhibiting intense green fluorescence with condensed chromatin distributed in a granular pattern (iii) Late apoptotic cells displaying orange-red fluorescence with nuclear fragmentation and apoptotic body formation and (iv) Rare necrotic cells identified by diffuse orange-red fluorescence with nuclear swelling. The predominant observation of late apoptotic cellular morphology, characterized by membrane blebbing, chromatin condensation and nuclear fragmentation with the development of membrane-enclosed apoptotic bodies in FeNP-treated cells relative to the predominantly viable green-fluorescing cells observed in untreated controls provides compelling evidence that S. molle-synthesized FeNPs induce programmed cell death through an apoptotic mechanism in HeLa cells. The elevated proportion of orange/red-fluorescing cells in treated samples relative to controls clearly demonstrates treatment-induced membrane permeabilization characteristic of the late apoptotic stage.
DISCUSSION
The present investigation provides comprehensive indication for the strong in vitro anticancer activity of iron nanoparticles synthesised by plant based from S. molle leaf aqueous extract against HeLa cells. The integration of physicochemical quantitative cytotoxicity cell characterization, morphological and fluorescence-based apoptosis finding collectively extended mechanistic framework for understanding the antiproliferative and pro-apoptotic effects of the synthesised nanomaterial. FeNP nanoparticles exposed potent antimicrobial possible against certain bacterial strains. In the same way, the amastigote and promastigote forms of the parasite Leishmania tropica were existing to be susceptible to the synthesized NPs. antioxidant, ABTS, α-amylase, α-glucosidase, acetylcholinesterase, and butyrylcholine esterase were potently inhibited by these nanoparticles, representative that they could be efficiently used as therapeutic agents for the oxidative stress and linked difficulties, such as diabetes and Alzheimer’s (Zafar et al., 2022). In the anticancer examination, the cells given with biosynthesized FeNPs were calculated by MTT assay for 2 days as respects the anti-Human Cholangiocarcinoma and Cytotoxic properties towards normal (HUVEC) and cholangiocarcinoma carcinoma cell lines, i.e. HCM-CSHL-0174-C22, CCLP-1, and QBC939. The IC50 values of biosynthesized FeNPs were 196, 237, and 278 µg/mL against HCM-CSHL-0174-C22, CCLP-1, and QBC939 cell lines, respectively. The viability of the cholangiocarcinoma cell line reduced dose-dependently in the occurrence of FeNPs (Zhao et al., 2022). S. molles are a high cause of secondary metabolites. 13 Gallo tannins, 3 phenolic acids, phenolic acid glucoside, 3 phenolic acid esters, organic acid, Gallo tannin derivative and 9 flavonoids were described and more quantified. Their study revealed that feruloyl tartaric A, a phenolic acid ester was the main phenolic acid while quercetin 3-O-glucuronide and catechin were the major flavonoids, and digalloylshikimic acid B as well as digalloylquinic acid were the leading tannins (Bvenura et al., 2022). No decrease at the 0.1 mg/mL concentration (n=9), the usual signal decrease at 0.5 mg/mL was 6.5 % (n=9, ±=8.0%), and the average signal reduction for the 1 mg/mL concentration was 11.12 % (n=9, ± =10.19%). There was no substantial change among the control cells and the 0.1 mg/mL, or 0.5 mg/mL test cells. The 1 mg/mL signal was found to be significant (p=0.0055). The correction factor determined was applied to the measured values using the formula (Martin et al., 2024). The maximum amount of late apoptotic cells was detected at the 9 µg/mL dose of CO-NPs (Cinchona officinalis extract-loaded iron oxide nanoparticles) when compared to COE (Cinchona Officinalis Extract). Physical counting of apoptotic and necrotic cells within an average of 300 cells presents with 83% in apoptotic and 6% were present with necrotic morphology (Al-Harbi et al., 2022).
CONCLUSION
The present investigation proves that iron nanoparticles plant-based synthesis from S. molle leaf aqueous extract exhibit significant and concentration-dependent anticancer activity against HeLa cells in vitro. FTIR spectroscopic characterization confirmed effective phytochemical capping and stabilization of synthesized FeNPs through O-H, C=O, and C-O functional group interactions. MTT cytotoxicity assay conventional an IC₅₀ of approximately 5 μg/mL against HeLa cells, with capability of declining progressively to 23.1±1.8% at 100 μg/mL. Phase contrast microscopy validated concentration reliant on morphological changes revealing of cytotoxic stress, whilst AO/EtBr fluorescence staining gives definitive evidence of apoptotic cell death as the initial cytotoxic mechanism, characterized by chromatin condensation, nuclear fragmentation and apoptotic body development. These results cooperatively found green based development of S. molle FeNPs as capable anticancer candidates warranting further mechanistic investigation. The exploitation of S. molle phytochemicals as green reducing and functionalizing agents represents a convergence of ethnobotanical knowledge and nanotechnology that may contribute to the growth of additional efficacious and smaller toxic anticancer therapeutic modalities.
