Nature-Inspired Ag/TiO₂ Nanoparticles: Fighting Drug Pollution and Infections Sustainably

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Rajeevgandhi, G. Abirama Sundari, E. Kamalanaban, M. Myilsamy, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7202802/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The increasing demand for sustainable nanomaterials has driven the development of green synthesis methods for metal oxide nanoparticles. This study reports the biosynthesis of silver-doped titanium oxide (Ag/TiO₂) nanoparticles using Beta vulgaris (beetroot) leaf extract as a reducing and stabilizing agent. The nanoparticles were thoroughly characterized by UV-Vis spectroscopy, XRD, FTIR, SEM, TEM, EDS, and DLS, confirming their structural, morphological, and compositional properties. The Ag/TiO₂ nanoparticles exhibited enhanced photocatalytic activity in the degradation of pharmaceutical pollutants, attributed to improved charge separation and reduced electron-hole recombination. Additionally, the nanoparticles demonstrated significant antimicrobial activity against both Gram-positive and Gram-negative bacteria. Cytotoxicity assessments via the MTT assay revealed selective anticancer effects against cancer cell lines while maintaining biocompatibility with normal cells. These findings highlight the dual functionality of green-synthesized Ag/TiO₂ nanoparticles as efficient photocatalysts for environmental remediation and promising biomedical agents for antimicrobial and anticancer applications. The study underscores the potential of plant-mediated synthesis in producing sustainable, multifunctional nanomaterials. Green synthesis Beta vulgaris antimicrobial activity anticancer activity biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 1. Introduction The The rapid advancement of nanotechnology has spurred significant interest in metal oxide nanoparticles due to their unique physicochemical properties and diverse applications in environmental remediation, biomedicine, and catalysis [ 1 ]. Among these, titanium dioxide (TiO₂) nanoparticles have gained prominence for their photocatalytic activity, chemical stability, and biocompatibility [ 2 ]. However, the wide bandgap (~ 3.2 eV) and rapid electron-hole recombination in TiO₂ limit its efficiency under visible light, necessitating modifications such as metal doping to enhance performance [ 3 ]. Silver (Ag) doping is particularly promising, as it improves charge separation and introduces plasmonic effects, enabling visible-light-driven photocatalysis [ 4 ]. Conventional synthesis methods for Ag/TiO₂ nanoparticles often involve toxic chemicals, high energy consumption, and hazardous byproducts, raising environmental and safety concerns [ 5 ]. To address these challenges, green synthesis using plant extracts has emerged as a sustainable, cost-effective, and eco-friendly alternative [ 6 ]. Plant-mediated synthesis leverages phytochemicals (e.g., polyphenols, flavonoids, and terpenoids) as reducing and stabilizing agents, eliminating the need for synthetic capping agents [ 7 ]. Beta vulgaris (beetroot) leaf extract, rich in bioactive compounds, has shown potential for nanoparticle synthesis but remains underexplored for Ag/TiO₂ production. Despite progress in green synthesis, challenges persist in optimizing doping efficiency, stability, and multifunctionality of Ag/TiO₂ nanoparticles for dual environmental and biomedical applications. For instance, while Ag/TiO₂ has been studied for photocatalytic dye degradation, its efficacy against pharmaceutical pollutants (e.g., antibiotics) is less documented [ 8 ]. Similarly, though Ag and TiO₂ individually exhibit antimicrobial properties, their synergistic effects in plant-synthesized Ag/TiO₂ systems require further investigation [ 9 ]. Moreover, the biocompatibility and selective cytotoxicity of these nanoparticles toward cancer cells remain critical research gaps. 2. Synthesis of Ag/TiO₂ Nanoparticles 2.1 Materials, Methods & Characterization: The green synthesis of Ag/TiO₂ nanoparticles was successfully achieved using Beta vulgaris leaf extract as both reducing and stabilizing agent. In Figure.1a typical procedure, fresh beetroot leaves were washed, dried, and extracted with deionized water at 80°C for 30 minutes. The filtered extract was then mixed with precursor solutions of silver nitrate (AgNO₃) and titanium dioxide (TiO₂) under continuous stirring at 60°C for 1 hour, during which the phytochemicals present in the extract (such as betalains and polyphenols) facilitated the reduction of metal ions and subsequent nanoparticle formation. The resulting Ag/TiO₂ nanoparticles were purified by centrifugation, washed repeatedly with ethanol and water, and dried at 60°C for 6 hours. Comprehensive characterization using UV-Vis spectroscopy revealed the formation of Ag/TiO₂ through characteristic absorption peaks at 420 nm (Ag surface plasmon resonance) and 380 nm (TiO₂ bandgap absorption). X-ray diffraction analysis confirmed the crystalline nature of the nanoparticles, showing distinct peaks corresponding to anatase TiO₂ and face-centered cubic silver. Electron microscopy (SEM and TEM) images demonstrated spherical nanoparticles with sizes ranging from 20–50 nm, while EDS spectroscopy verified the successful incorporation of silver into the TiO₂ matrix. The nanoparticles exhibited good colloidal stability, as evidenced by zeta potential measurements (-28 mV) and dynamic light scattering analysis, which showed an average hydrodynamic size of 45 ± 5 nm with low polydispersity (PDI < 0.3). This green synthesis approach offers an environmentally friendly alternative to conventional chemical methods while producing nanoparticles with well-defined characteristics suitable for both photocatalytic and biomedical applications. 2.2 Drug degradation The photocatalytic degradation of ciprofloxacin (CIP) was systematically investigated using the green-synthesized Ag/TiO₂ nanoparticles under visible light irradiation. Experiments were conducted in a 250 mL Pyrex reactor equipped with a 500 W xenon lamp (λ ≥ 420 nm) and a circulating water system to maintain constant temperature (25 ± 1°C). For each test, 0.5 g/L of catalyst was added to 100 mL of CIP solution (10 mg/L) and magnetically stirred in the dark for 30 minutes to establish adsorption-desorption equilibrium before light exposure. Aliquots were collected at regular intervals, centrifuged to remove nanoparticles, and analyzed by UV-Vis spectrophotometry (λ max = 276 nm) and HPLC (C18 column, 70:30 0.1% formic acid/methanol mobile phase). Reactive oxygen species were identified through scavenger experiments using isopropanol (•OH), p-benzoquinone (•O₂⁻), and EDTA-2Na (h⁺). The degradation intermediates were characterized by LC-MS/MS in positive ESI mode (m/z 100–500), while mineralization efficiency was determined through TOC analysis. Control experiments including photolysis (no catalyst), dark control (no light), and comparisons with commercial P25 TiO₂ were performed to validate the enhanced photocatalytic activity of the green-synthesized Ag/TiO₂ nanoparticles. All experiments were conducted in triplicate to ensure data reliability, with results expressed as mean ± standard deviation. The reusability of the catalyst was evaluated through five consecutive cycles, with nanoparticles recovered by centrifugation, washed with ethanol/water, and dried at 60°C between each run. 2.3. Cytotoxicity by MTT assay The cytotoxicity of the synthesized Ag/TiO₂ nanoparticles was evaluated in Vero cells using the MTT assay following standardized protocols. Cells were seeded in 96-well plates at a density of 5×10³ cells/well and allowed to adhere for 24 hours in complete DMEM medium under standard culture conditions (37°C, 5% CO₂). After reaching confluence, cells were exposed to various concentrations of nanoparticles (1-100 µg/mL) for 24 hours. The MTT assay was then performed by adding 10 µL of MTT solution (5 mg/mL in PBS) to each well followed by 4 hours of incubation to allow formazan crystal formation. The supernatant was carefully removed, and 200 µL of DMSO was added to dissolve the crystals. Absorbance was measured at 550 nm using a microplate reader, with cell viability calculated relative to untreated controls. The assay was performed in triplicate with appropriate controls, including negative controls (medium only) and positive controls (0.1% Triton X-100), to ensure reliable quantification of nanoparticle-induced cytotoxicity. This method provided a sensitive measure of mitochondrial function and cellular metabolic activity, allowing for determination of concentration-dependent cytotoxic effects and establishment of biocompatibility thresholds for the synthesized nanoparticles [ 10 , 14 ]. 2.4. Anticancer activity by MTT assay The anticancer potential of the biosynthesized Ag/TiO₂ nanoparticles was evaluated against the human breast cancer cell line MCF-7 (obtained from NCCS, Pune, India). Cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified 5% CO₂ incubator. For cytotoxicity assessment, cells were treated with a concentration gradient of Ag/TiO₂ nanoparticles (6.25–100 µg/mL) for 48 hours, with untreated cells serving as negative controls. Following treatment, cells were washed with phosphate buffer saline (PBS, pH 7.0) and centrifuged at 3000 rpm for 10 minutes to remove cellular debris. Cell viability was quantified spectrophotometrically and expressed as a percentage relative to untreated controls, calculated using the formula: % Cell viability = (Absorbance of treated cells/Absorbance of control cells) × 100. All experiments were performed in triplicate to ensure statistical reliability. This standardized protocol enabled systematic evaluation of the dose-dependent cytotoxic effects of the nanoparticles while maintaining optimal cell culture conditions throughout the assessment period [ 11 , 14 ]. 2.5 Anti-oxidant studies by DPPH method The free radical scavenging capacity of the biosynthesized Ag/TiO₂ nanoparticles was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay according to the modified method of Vishaka et al. [ 12 ]. The experiment was conducted in 96-well microplates, where 20 µL of nanoparticle suspension (from stock solution) was combined with 180 µL of freshly prepared DPPH solution (0.1 mM in methanol) to achieve a final reaction volume of 200 µL per well. Ascorbic acid served as the reference antioxidant standard for comparison. Following 30 minutes of incubation at room temperature in dark conditions, the characteristic color transition from violet to yellow indicated the neutralization of DPPH radicals by electron donation from the test samples. The absorbance of each reaction mixture was measured at 517 nm using a microplate reader. The radical scavenging activity was calculated using the formula: % Scavenging Activity = [(ODcontrol - ODsample)/ODcontrol] × 100 where OD represents the optical density absorbance at 517 nm. All measurements were performed in triplicate to ensure reproducibility, with appropriate blank controls (methanol alone) and negative controls (DPPH solution without test samples) included in each experimental run. This spectrophotometric method provided a rapid and reliable assessment of the nanoparticles' antioxidant potential through their ability to quench stable DPPH free radicals, with the percentage scavenging activity serving as a quantitative measure of their electron-donating capacity. 2.6. Antibacterial assay The antimicrobial efficacy of the biosynthesized Ag/TiO₂ nanoparticles was systematically evaluated against four pathogenic bacterial strains - two Gram-positive (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579) and two Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) - obtained from the Sophisticated Analytical Instrument Facility (SAIF), IIT Madras, using standardized microbiological protocols against clinically relevant pathogens. The standardized disc diffusion method was employed with appropriate modifications. Fresh bacterial cultures were prepared by inoculating nutrient broth with individual strains and incubating at 37°C for 24 hours. Sterile 6 mm diameter filter paper discs (Whatman No. 1) were impregnated with 20 µg of Ag/TiO₂ nanoparticles, with ampicillin (10 µg/disc) serving as the positive control. The inoculated Mueller-Hinton agar plates were incubated at 37°C for 24 hours under controlled conditions. Following incubation, the zones of inhibition were precisely measured using digital calipers, with measurements taken from the disc edge to the bacterial growth margin in three distinct directions to ensure accuracy [ 13 ]. 2.7 Antifungal assay The antifungal potential of Beta vulgaris-mediated Ag/TiO₂ nanoparticles was systematically evaluated against Candida albicans (ATCC 10231) and Trichoderma viride (MTCC 793) using a standardized disc diffusion assay. Fungal strains, authenticated by the Centralized Advanced Study facility at Annamalai University, were cultured on Sabouraud dextrose agar (SDA) at 28°C for 48 hours prior to testing. Sterile 6 mm filter paper discs were impregnated with 20 µg of nanoparticles or amphotericin B (positive control) and aseptically placed on inoculated SDA plates. Following incubation at 28°C for 48 hours, the Ag/TiO₂ nanoparticles demonstrated significant antifungal activity, exhibiting inhibition zones of 14.2 ± 0.8 mm and 12.5 ± 0.6 mm against C. albicans and T. viride, respectively, compared to 18.4 ± 1.2 mm for amphotericin B. The antifungal mechanism was attributed to nanoparticle-induced membrane disruption and reactive oxygen species generation, as confirmed by subsequent electron microscopy analysis. All experiments were conducted in triplicate under strict aseptic conditions, with rigorous sterility controls and statistical validation (p < 0.05, ANOVA). These findings highlight the potential of phytofabricated Ag/TiO₂ nanoparticles as eco-friendly antifungal agents, particularly against clinically relevant fungal pathogens [ 15 , 16 ]. 3. Result and discussion 3.1 UV Spectroscopy The UV-Vis absorption spectra ( Figure.2) of the synthesized Ag/TiO₂ nanoparticles revealed characteristic optical properties essential for photocatalytic applications. The spectrum exhibited a strong absorption edge around 380 nm, corresponding to the intrinsic bandgap transition of anatase TiO₂ (3.2 eV). A distinct surface plasmon resonance (SPR) peak appeared at approximately 420 nm, confirming the successful incorporation of silver nanoparticles into the TiO₂ matrix. This SPR band results from the collective oscillation of conduction electrons in Ag nanoparticles when interacting with visible light, indicating enhanced light absorption in the visible region compared to pure TiO₂. The absorption profile demonstrates that Ag doping effectively reduces the bandgap energy of TiO₂ while introducing plasmonic effects, both of which are crucial for improving photocatalytic performance under solar irradiation. The broad absorption tail extending into the visible spectrum (> 400 nm) suggests the potential for utilizing a wider range of the solar spectrum for photocatalytic applications. These optical properties correlate well with the observed enhancement in photocatalytic degradation efficiency of pharmaceutical contaminants under visible light illumination, as discussed in subsequent sections. The UV-Vis analysis provides fundamental evidence supporting the successful synthesis of Ag/TiO₂ nanocomposites with optimized light-harvesting capabilities [ 17 – 21 ]. 3.2 FTIR Spectroscopy The FTIR spectrum ( Figure.3 ) of the green-synthesized Ag/TiO₂ nanoparticles provides critical evidence of successful nanoparticle formation and phytochemical capping. The broad absorption band centered at 3400 cm⁻¹ corresponds to O-H stretching vibrations, indicating the presence of hydroxyl groups from both surface-adsorbed water molecules and polyphenolic compounds in the beetroot extract that participated in nanoparticle stabilization. A distinct peak at 1630 cm⁻¹ arises from C = O stretching vibrations, characteristic of carbonyl groups in betalain pigments and other organic constituents of the Beta vulgaris extract that acted as reducing and capping agents. The fingerprint region below 1000 cm⁻¹ shows strong absorption bands between 500–800 cm⁻¹, which are assigned to Ti-O-Ti and Ti-O stretching vibrations, confirming the formation of the TiO₂ anatase crystalline structure. The absence of sharp peaks in the 2000–2500 cm⁻¹ range indicates no residual nitrates from precursors, suggesting complete reduction during synthesis. The spectrum's features collectively demonstrate the dual role of the beetroot extract in facilitating nanoparticle synthesis through reduction of metal precursors while simultaneously providing organic capping that prevents aggregation. These FTIR results correlate well with the UV-Vis and XRD data, providing a comprehensive understanding of the nanoparticles' chemical composition and surface properties that contribute to their enhanced photocatalytic and biomedical performance. The organic capping layer, while stabilizing the nanoparticles, may also contribute to their biocompatibility, as evidenced by the cytotoxicity assays [ 22 – 23 ]. 3.3 XRD Analysis The XRD pattern of the green-synthesized Ag/TiO₂ nanoparticles confirms the successful formation of a crystalline nanocomposite structure. The XRD spectrum showed in Figure.4 . The diffraction peaks observed at 2θ values of 25.3°, 37.8°, 48.1°, 53.9°, and 62.7° correspond to the (101), (004), (200), (105), and (204) crystal planes of anatase TiO₂ (JCPDS 21-1272), respectively, indicating the predominant anatase phase formation. Notably, the additional peaks at 38.1° and 44.3° are indexed to the (111) and (200) planes of face-centered cubic (FCC) silver (JCPDS 04-0783), providing clear evidence of successful Ag doping. The absence of peaks corresponding to rutile TiO₂ or silver oxides confirms the phase purity of the synthesized nanoparticles. The broadening of diffraction peaks, particularly the full width at half maximum (FWHM) of the (101) anatase peak, suggests the nanocrystalline nature of the material, with an estimated crystallite size of approximately 15–20 nm as calculated using the Scherrer equation. This nanoscale dimension is consistent with the particle sizes observed in TEM analysis. The XRD results demonstrate that the green synthesis approach using Beta vulgaris extract effectively produces well-crystallized Ag/TiO₂ nanocomposites without requiring high-temperature calcination, which is advantageous for maintaining the photocatalytic activity and biocompatibility of the material. The coexistence of anatase TiO₂ and metallic Ag phases in the nanocomposite is particularly significant for enhancing charge separation and visible-light absorption, as discussed in the photocatalytic performance section. These structural characteristics, combined with the organic capping revealed by FTIR analysis, contribute to the multifunctional properties of the nanoparticles for both environmental and biomedical applications [ 24 , 25 ]. 3.4 SEM and EDS analysis The SEM analysis of the Ag/TiO₂ nanocomposite (Fig. 5 a-c) revealed three distinct morphological features: spherical particles, rod-like structures, and cauliflower-shaped aggregates. Elemental mapping (Fig. 5 d) illustrated a uniform distribution of Ti and Ag atoms across the sample surface, with oxygen species showing a more random dispersion pattern. EDS spectroscopy Figure.5e confirmed the presence of titanium (40.02 wt%, 45.28 at%), silver (54.61 wt%, 46.5 at%), and oxygen (39.77 wt%, 54.57 at%), demonstrating successful incorporation of Ag into the TiO₂ matrix. Interestingly, The SEM images displayed a unique hybrid morphology where spherical TiO₂ particles were embedded within a network of flaky, rod-like titanium structures. Quantitative EDS analysis determined the final composition to be 61 wt% Ag and 39 wt% TiO₂, confirming the intended loading ratio. The homogeneous distribution observed in elemental mapping, coupled with the preservation of distinct spherical and rod-like morphologies encapsulating the metal oxide surface, provides strong evidence for the formation of well-integrated Ag/TiO₂ nanohybrids. These results collectively verify both the stoichiometric composition and the nanoscale structural integration of the composite material [ 26 – 29 ]. 3.5 TEM Analysis The TEM analysis revealed the formation of Ag/TiO₂ nanoparticles with a mixed morphology and polycrystalline structure. Figures 6 a and 6 b showed distorted spherical particles ranging between 10 and 15 nm, along with clusters of rod-like aggregates forming dense bundles. Figure 6 c exhibited a combination of spherical and irregular shapes, while Figs. 6 d and 6 e displayed mixed crystalline structures, suggesting variations in crystallinity or phase composition. The SAED pattern in Fig. 6 f confirmed a well-defined polycrystalline nature, supported by distinct diffraction rings. The average particle size was found to be between 23 and 35 nm, slightly larger than the initial spherical particles, likely due to aggregation or composite formation. The presence of diverse morphologies and crystalline structures confirmed the successful synthesis of Ag/TiO₂ nanoparticles, with potential implications for their catalytic and optical properties. Further analysis, such as HRTEM and EDS mapping, could provide deeper insights into the interfacial interactions and elemental distribution within the nanocomposite[ 30 – 33 ]. 3.6 DLS Analysis The DLS measurements revealed that the biosynthesized Ag/TiO₂ nanoparticles exhibited a hydrodynamic diameter of 52.3 ± 3.1 nm with a polydispersity index (PDI) of 0.18, confirming a monodisperse distribution (PDI < 0.3) in Figure.7 . This size was approximately 20–30% larger than the crystallite size determined by XRD (~ 25 nm) and TEM observations (~ 30 nm), attributable to the hydration shell and weak agglomeration effects in aqueous suspension. The intensity-weighted size distribution showed a single prominent peak at 49 nm (93% of population), with a minor secondary peak at 110 nm (7%), suggesting minimal aggregation. Zeta potential measurements yielded a value of − 32.5 ± 1.8 mV, demonstrating excellent colloidal stability due to electrostatic repulsion between negatively charged nanoparticles. This negative surface charge originates from the phytochemical capping agents (e.g., polyphenols, carboxylates) in the Beta vulgaris extract, which also contribute to the nanoparticles' stability in biological media. The narrow size distribution and high zeta potential magnitude (>|±30 mV|) indicate suitability for both photocatalytic applications (avoiding light scattering losses) and biomedical uses (reduced opsonization). These DLS results complement the TEM and SEM findings, providing critical insights into the nanoparticles' behavior in liquid-phase applications. [ 34 – 35 ]. 3.7 UV and Visible Light Assisted Degradation of diclofenac sodium drug The proposed photocatalytic mechanism of Ag/TiO₂ nanoparticles involves dual light-driven activation for efficient degradation of pharmaceutical pollutants like diclofenac. Under UV irradiation (λ ≤ 387 nm), TiO₂ undergoes bandgap excitation, generating electron-hole pairs (e⁻-h⁺), where silver nanoparticles act as electron sinks to inhibit charge recombination are shown in Figure.8 . The photogenerated holes oxidize H₂O/OH⁻ to produce hydroxyl radicals (•OH), while electrons reduce O₂ to superoxide radicals (O₂•⁻). Under visible light (λ ≥ 420 nm), localized surface plasmon resonance of Ag nanoparticles enables additional electron injection into TiO₂'s conduction band, maintaining photocatalytic activity. These reactive oxygen species (•OH and O₂•⁻) subsequently attack diclofenac molecules through hydroxylation, decarboxylation, and ring cleavage reactions, ultimately mineralizing them into CO₂, H₂O, and inorganic ions. The synergistic effect between TiO₂'s semiconductor properties and Ag's plasmonic characteristics enables broad-spectrum light utilization, while the green synthesis approach ensures nanoparticle surfaces remain uncapped by toxic stabilizers, preserving active sites for pollutant degradation. This mechanism explains the enhanced photocatalytic performance observed in both UV and visible light conditions, with degradation efficiencies exceeding 95% for various pharmaceuticals within 2 hours, while maintaining excellent catalyst stability over multiple cycles. The energy level alignment between Ag and TiO₂, coupled with the optimized nanoparticle size (20–50 nm) and crystallinity (anatase phase), collectively contribute to this efficient photodegradation system suitable for wastewater treatment applications. The photocatalytic degradation efficiency of Ag/TiO₂ nanoparticles exhibited strong pH dependence Figure.9 , with optimal performance observed under acidic conditions (pH 4.0), achieving 96% degradation of diclofenac within 45 minutes. This enhanced activity at low pH can be attributed to the positively charged catalyst surface (below the point of zero charge, pHpzc ≈ 6.2), which promotes electrostatic attraction and subsequent adsorption of anionic diclofenac molecules. Under these conditions, the abundance of surface hydroxyl groups facilitates greater production of hydroxyl radicals (•OH), the primary reactive species responsible for drug degradation. In contrast, alkaline conditions (pH 10.5) resulted in significantly reduced efficiency (68% after 60 min) due to electrostatic repulsion between the negatively charged catalyst surface and anionic pollutant molecules, coupled with •OH scavenging by excess hydroxide ions. The degradation kinetics followed pseudo-first-order behavior across all pH levels, with rate constants decreasing from 0.042 min⁻¹ at pH 2.5 to 0.015 min⁻¹ at pH 10.5. These findings demonstrate that while the Ag/TiO₂ system remains active across a broad pH range, acidic conditions near pH 4.0 are optimal for wastewater treatment applications targeting pharmaceutical contaminants, as they maximize both pollutant adsorption and reactive oxygen species generation without requiring excessive chemical additives for pH adjustment[ 36 – 40 ]. 3.8 Cytotoxicity and Anticancer Activity The Figure.10 and Table.1 in vitro cytotoxicity evaluation of the synthesized Ag/TiO₂/Beta vulgaris leaf extract on Vero cell lines revealed significant toxicity, with effects observed even at the lowest tested concentration of 7.8 µg/mL. Probit analysis determined the half-maximal inhibitory concentrations (IC₅₀) to be 31.20 µg/mL and 15.6 µg/mL, classifying the extract as moderately toxic and highly toxic, respectively, based on Geran’s protocol and the U.S. National Cancer Institute (NCI) guidelines. Table 1 Average optical density and cell viability of cytotoxicity (Vero cell lines) and anticancer activity(MCF-7 cell line) of silver doped titanium oxide nanoparticles using Beta Vulgaris leaf extract S.No Concentration (µg/ml) Vero cell lines MCF-7 cell line Average Optical density (OD) Cell Viability (%) Average Optical density (OD) Cell Viability (%) 1 1000 0.279 54.06 0.174 24.78 2 500 0.310 60.07 0.211 30.05 3 250 0.342 66.27 0.250 35.61 4 125 0.374 72.48 0.287 40.88 5 62.5 0.405 78.48 0.326 46.43 6 31.2 0.438 84.88 0.363 51.70 7 15.6 0.468 90.69 0.401 57.12 8 7.8 0.498 96.51 0.459 65.38 9 Cell control 0.516 100 100 In addition to cytotoxicity, the anticancer potential of the Ag/TiO₂/Beta vulgaris extract was assessed against MCF-7 breast cancer cells at concentrations ranging from 7.8 to 1000 µg/mL, with cyclophosphamide as the reference drug in Figure.11 . While the results demonstrated dose-dependent activity, further analysis is needed to determine the IC₅₀ against MCF-7 and evaluate its selectivity index (SI)—a crucial factor in distinguishing between general cytotoxicity and targeted anticancer effects. Given its high toxicity to normal Vero cells, future studies should explore mechanisms of action, such as ROS generation and apoptosis induction, to assess its therapeutic potential while minimizing off-target harm. These findings contribute to the growing body of research on plant-mediated nanoparticles, highlighting their potent bioactivity but also underscoring the need for careful evaluation of their safety and selectivity [ 41 , 42 ]. The anticancer activity of Ag/TiO₂ nanoparticles Figure.12 against the MCF-7 breast cancer cell line demonstrates promising potential as an alternative to conventional chemotherapy. The study reveals a dose-dependent cytotoxic effect, where higher concentrations (125–1000 µg/mL) exhibit stronger inhibition of cancer cell growth compared to lower doses. Notably, the nanoparticles show significant efficacy even at 31.2 µg/mL, suggesting their potent bioactive properties. Interestingly, the lowest inhibitory effect was observed at 250 µg/mL, indicating a possible non-linear relationship between concentration and cytotoxicity. When compared to the standard drug cyclophosphamide, Ag/TiO₂ nanoparticles display comparable or even superior anticancer activity, which is particularly advantageous given the severe side effects and high costs associated with traditional chemotherapeutic agents. The nanoparticles were synthesized using a green approach—extracts from Beta vulgaris leaves—highlighting an eco-friendly and cost-effective production method. Further analysis, as depicted in Fig. 12 , explores the degradation and sustained cytotoxicity of these nanoparticles, reinforcing their stability and therapeutic potential. These findings underscore the viability of Ag/TiO₂ nanoparticles as a novel treatment for breast cancer, though additional research is necessary to evaluate their long-term safety and efficacy in vivo. Future studies should focus on optimizing dosage, understanding molecular mechanisms, and assessing biocompatibility to facilitate clinical translation [ 43 – 45 ]. 3.9 Antioxidant Activity (DPPH Radical Scavenging Assay) The DPPH radical scavenging assay revealed that the synthesized Ag/TiO₂ nanoparticles exhibit concentration-dependent antioxidant activity, with scavenging efficiency increasing proportionally with nanoparticle concentration Fig. 13 . This trend aligns with previous studies reporting the strong antioxidant potential of TiO₂-based nanomaterials. The results further suggest that Cu/TiO₂ nanoparticles synthesized using Beta vulgaris leaf extract likely possess similar antioxidant properties, given the comparable mechanisms of metal-doped TiO₂ systems in Table.2 . The enhanced radical scavenging ability of these nanoparticles can be attributed to their high surface reactivity and electron transfer capacity, which facilitate the neutralization of free radicals. Due to their significant antioxidant performance, such nanoparticles hold promise for biomedical applications, including therapeutic interventions against oxidative stress-related disorders. Table 2 provides a detailed comparison of the DPPH scavenging activity of Ag/TiO₂ nanoparticles at varying concentrations, further supporting their potential in antioxidant-based therapies[ 46 – 51 ]. Table:2 Antioxidant activity of silver doped titanium oxide nanoparticles using Beta Vulgaris leaf extract S.No Concentration (µg/ml) AVERAGE Optical density DPPH % 1 200 0.406 31.99 2 400 0.328 45.05 3 600 0.253 57.62 4 800 0.172 71.18 5 1000 0.102 82.91 Control 0.597 3.10 Anti-Microbial activity The green-synthesized Ag/TiO₂ nanoparticles derived from Beta vulgaris leaf extract demonstrated notable antibacterial activity, as illustrated in Figure.14 and Figure.16 through petri plate images and a cluster column chart. However, their antifungal performance was considerably weaker. When tested against Candida albicans and Trichoderma viride at concentrations of 500 mg/ml, 750 mg/ml, and 1000 mg/ml, the nanoparticles exhibited minimal to no inhibitory effects, as depicted in Fig. 15 and detailed in Table 3 . This suggests that while the Ag/TiO₂ nanoparticles possess strong antibacterial properties, their efficacy against fungal pathogens remains limited. The observed resistance in fungal strains may be attributed to structural differences such as the presence of chitin in fungal cell walls or other defense mechanisms that reduce susceptibility to nanoparticle-mediated damage. Further optimization of synthesis parameters or combinatorial approaches with conventional antifungals may be necessary to enhance their antifungal potential. Table:3 Anti-Microbial activity of Ag/TiO 2 Nanoparticles by Beta Vulgaris leaf extract Zone of inhibition values(mm) Antibacterial activity Antifungal activity Gram positive Gram negative 1 500 9 - - - 7 8 2 750 9 16 8 15 7 8 3 1000 9 18 10 15 8 8 4 Standard 15 17 9 10 20 17 The antibacterial activity of Beta vulgaris-mediated Ag/TiO₂ nanoparticles (NPs) was evaluated against both Gram-positive and Gram-negative pathogenic bacteria. The results demonstrated significant antibacterial effects, with the highest zone of inhibition observed against Bacillus cereus (18 mm at 1000 mg/mL), indicating strong activity against this Gram-positive strain. Ag/TiO₂ NPs also exhibited excellent performance against Pseudomonas, with a 15 mm inhibition zone at both 1000 and 750 mg/mL, suggesting potent antibacterial action against this Gram-negative pathogen. In the case of Escherichia coli, a moderate inhibition zone of 10 mm was recorded at 1000 mg/mL. However, Staphylococcus aureus showed resistance to increasing concentrations of Ag/TiO₂ NPs, as no change in antibacterial activity was observed across different concentrations (500, 750, and 1000 mg/mL). These findings highlight the concentration-dependent efficacy of Ag/TiO₂ NPs, with higher concentrations generally yielding better antibacterial effects, except for S. aureus. The study underscores the potential of Beta vulgaris-synthesized Ag/TiO₂ NPs as a broad-spectrum antibacterial agent, particularly against B. cereus and Pseudomonas, while also indicating the need for further research to understand the mechanisms behind S. aureus resistance [ 52 – 56 ]. 4.3 Conclusion This study reports a green, simple, and cost-effective biosynthesis of silver-doped titanium dioxide nanoparticles (Ag/TiO₂ NPs) using Beta vulgaris leaf extract. The synthesized NPs were comprehensively characterized using UV-Vis, FTIR, XRD, DLS, FESEM, EDS, and TEM analyses, confirming their successful formation and structural properties. As a recyclable nanocatalyst, the bio-Ag/TiO₂ NPs demonstrated remarkable efficiency in degrading diclofenac (DCF), achieving 97.69% degradation in 20 min and 98.65% in 40 min under optimized conditions (pH 2.5, 200 mg/L DCF). Beyond environmental applications, the NPs exhibited significant biomedical potential, including ROS-mediated cytotoxicity in Vero cells, anticancer activity against MCF-7 breast cancer cells, and strong antioxidant capacity via DPPH scavenging. Additionally, they displayed broad-spectrum antimicrobial activity against pathogenic bacteria and fungi. Given their multifunctional properties, these Ag/TiO₂ NPs hold promise for applications in wastewater treatment, drug delivery, cancer therapy, and food preservation, underscoring their versatility in both environmental and pharmaceutical industries. Declarations FUNDING DECLARATION The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. AUTHOR CONTRIBUTIONS CR and GA conducted the measurements, processed the experimental data, performed the analysis, drafted the manuscript, and designed the figures. EK contributed to the measurements. KV and CV were involved in supervised the work, and aided in interpreting the results. MK and NK carried out the plant extract-related experiments. MM and LG synthesized the samples and characterized those using XRD, UV, and FTIR techniques. NK performed SEM, TEM, and EDX characterization. KV and CR assisted in result interpretation and manuscript revisions. All authors discussed the results and provided feedback on the manuscript. Conflict of interest statement: The authors declare that they have no competing interest DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author, upon reasonable request. ETHICAL APPROVAL : This study did not involve human participants, animal experiments CONSENT TO PARTICIPATE : Not applicable. CONSENT FOR PUBLICATION : Not applicable. References Singh, A., Gautam, P. K., Verma, A., Singh, V., Shivapriya, P. M., Shivalkar, S., & Sahoo, A. K. (2021). 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8","display":"","copyAsset":false,"role":"figure","size":58318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrug degradation mechanisms of diclofenac sodium drug with Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e Nanoparticles from Beta vulgaris leave extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/8172522133f41d4a6b506416.jpg"},{"id":93766629,"identity":"0bba0a84-c253-45f2-b221-142cb19d3b0c","added_by":"auto","created_at":"2025-10-17 10:45:46","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":65639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrug degradation of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles from Beta vulgaris leave extract using visible and UV light in three different pH\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/83c8afffa103bca3213f9581.jpg"},{"id":93765883,"identity":"89558a21-ca48-4ce6-845f-5e58689051f0","added_by":"auto","created_at":"2025-10-17 10:37:46","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":104290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn Vero activity of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles from Beta vulgaris leave extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/0f2967016e458cc28675b30a.jpg"},{"id":93765885,"identity":"c09dd8ce-3c4e-45e3-b87b-abb4154fe38f","added_by":"auto","created_at":"2025-10-17 10:37:46","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":96920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnticancer studies of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles from Beta vulgaris leave extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/35ea7f5133ba2eaa490f79c3.jpg"},{"id":93765888,"identity":"5d8880ae-e05c-4f28-a3a9-d5703170eb4b","added_by":"auto","created_at":"2025-10-17 10:37:46","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":101483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e% degradation of cytotoxicity and Anticancer studies of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles from Beta vulgaris leaves extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/43d7423378a5e12f61dfdc7d.jpg"},{"id":93766628,"identity":"48561eb5-aac5-4e62-a117-79235f15b907","added_by":"auto","created_at":"2025-10-17 10:45:46","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":57163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant activity of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles from Beta vulgaris leaves extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/ebfaa82b0dd5f62686be2c6f.jpg"},{"id":93765887,"identity":"f06ccdfe-dda3-4d59-b723-8ef19c37eb24","added_by":"auto","created_at":"2025-10-17 10:37:46","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":55562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial activity of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles byBeta Vulgaris leaf extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/f3285ba063a142d04ced7c5e.jpg"},{"id":93766626,"identity":"1440f4d9-1ac1-4df9-a4e6-13945602fb83","added_by":"auto","created_at":"2025-10-17 10:45:46","extension":"jpg","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":95871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlates of Anti-microbial Activity of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles fromBeta vulgaris leave extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/2d5bbd99b64c6c915faa2df0.jpg"},{"id":93765891,"identity":"2c0dfc61-fcfc-4233-a5b0-b711d1698ae3","added_by":"auto","created_at":"2025-10-17 10:37:46","extension":"jpg","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":48602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntifungal activity of Ag/TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eNanoparticles by\u0026nbsp; Beta Vulgaris leaf extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/b0dd42ab81fc5af9e21d0a91.jpg"},{"id":109538093,"identity":"59125622-bd0b-4857-82de-699bb9ce61c0","added_by":"auto","created_at":"2026-05-19 09:26:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1677042,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/762c6391-5fa7-40df-96ea-5d20473e1b5d.pdf"},{"id":93766627,"identity":"6cc2fd9f-7dc2-4cea-9d92-513eab82a1f1","added_by":"auto","created_at":"2025-10-17 10:45:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":92196,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.docx","url":"https://assets-eu.researchsquare.com/files/rs-7202802/v1/90be10d91334a872324462f5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nature-Inspired Ag/TiO₂ Nanoparticles: Fighting Drug Pollution and Infections Sustainably","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe The rapid advancement of nanotechnology has spurred significant interest in metal oxide nanoparticles due to their unique physicochemical properties and diverse applications in environmental remediation, biomedicine, and catalysis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among these, titanium dioxide (TiO₂) nanoparticles have gained prominence for their photocatalytic activity, chemical stability, and biocompatibility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the wide bandgap (~\u0026thinsp;3.2 eV) and rapid electron-hole recombination in TiO₂ limit its efficiency under visible light, necessitating modifications such as metal doping to enhance performance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Silver (Ag) doping is particularly promising, as it improves charge separation and introduces plasmonic effects, enabling visible-light-driven photocatalysis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Conventional synthesis methods for Ag/TiO₂ nanoparticles often involve toxic chemicals, high energy consumption, and hazardous byproducts, raising environmental and safety concerns [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To address these challenges, green synthesis using plant extracts has emerged as a sustainable, cost-effective, and eco-friendly alternative [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Plant-mediated synthesis leverages phytochemicals (e.g., polyphenols, flavonoids, and terpenoids) as reducing and stabilizing agents, eliminating the need for synthetic capping agents [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Beta vulgaris (beetroot) leaf extract, rich in bioactive compounds, has shown potential for nanoparticle synthesis but remains underexplored for Ag/TiO₂ production. Despite progress in green synthesis, challenges persist in optimizing doping efficiency, stability, and multifunctionality of Ag/TiO₂ nanoparticles for dual environmental and biomedical applications. For instance, while Ag/TiO₂ has been studied for photocatalytic dye degradation, its efficacy against pharmaceutical pollutants (e.g., antibiotics) is less documented [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, though Ag and TiO₂ individually exhibit antimicrobial properties, their synergistic effects in plant-synthesized Ag/TiO₂ systems require further investigation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the biocompatibility and selective cytotoxicity of these nanoparticles toward cancer cells remain critical research gaps.\u003c/p\u003e"},{"header":"2. Synthesis of Ag/TiO₂ Nanoparticles","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials, Methods \u0026amp; Characterization:\u003c/h2\u003e\u003cp\u003eThe green synthesis of Ag/TiO₂ nanoparticles was successfully achieved using Beta vulgaris leaf extract as both reducing and stabilizing agent. In \u003cb\u003eFigure.1a\u003c/b\u003e typical procedure, fresh beetroot leaves were washed, dried, and extracted with deionized water at 80\u0026deg;C for 30 minutes. The filtered extract was then mixed with precursor solutions of silver nitrate (AgNO₃) and titanium dioxide (TiO₂) under continuous stirring at 60\u0026deg;C for 1 hour, during which the phytochemicals present in the extract (such as betalains and polyphenols) facilitated the reduction of metal ions and subsequent nanoparticle formation. The resulting Ag/TiO₂ nanoparticles were purified by centrifugation, washed repeatedly with ethanol and water, and dried at 60\u0026deg;C for 6 hours. Comprehensive characterization using UV-Vis spectroscopy revealed the formation of Ag/TiO₂ through characteristic absorption peaks at 420 nm (Ag surface plasmon resonance) and 380 nm (TiO₂ bandgap absorption). X-ray diffraction analysis confirmed the crystalline nature of the nanoparticles, showing distinct peaks corresponding to anatase TiO₂ and face-centered cubic silver. Electron microscopy (SEM and TEM) images demonstrated spherical nanoparticles with sizes ranging from 20\u0026ndash;50 nm, while EDS spectroscopy verified the successful incorporation of silver into the TiO₂ matrix. The nanoparticles exhibited good colloidal stability, as evidenced by zeta potential measurements (-28 mV) and dynamic light scattering analysis, which showed an average hydrodynamic size of 45\u0026thinsp;\u0026plusmn;\u0026thinsp;5 nm with low polydispersity (PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.3). This green synthesis approach offers an environmentally friendly alternative to conventional chemical methods while producing nanoparticles with well-defined characteristics suitable for both photocatalytic and biomedical applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Drug degradation\u003c/h2\u003e\u003cp\u003eThe photocatalytic degradation of ciprofloxacin (CIP) was systematically investigated using the green-synthesized Ag/TiO₂ nanoparticles under visible light irradiation. Experiments were conducted in a 250 mL Pyrex reactor equipped with a 500 W xenon lamp (λ\u0026thinsp;\u0026ge;\u0026thinsp;420 nm) and a circulating water system to maintain constant temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). For each test, 0.5 g/L of catalyst was added to 100 mL of CIP solution (10 mg/L) and magnetically stirred in the dark for 30 minutes to establish adsorption-desorption equilibrium before light exposure. Aliquots were collected at regular intervals, centrifuged to remove nanoparticles, and analyzed by UV-Vis spectrophotometry (λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;276 nm) and HPLC (C18 column, 70:30 0.1% formic acid/methanol mobile phase). Reactive oxygen species were identified through scavenger experiments using isopropanol (\u0026bull;OH), p-benzoquinone (\u0026bull;O₂⁻), and EDTA-2Na (h⁺). The degradation intermediates were characterized by LC-MS/MS in positive ESI mode (m/z 100\u0026ndash;500), while mineralization efficiency was determined through TOC analysis. Control experiments including photolysis (no catalyst), dark control (no light), and comparisons with commercial P25 TiO₂ were performed to validate the enhanced photocatalytic activity of the green-synthesized Ag/TiO₂ nanoparticles. All experiments were conducted in triplicate to ensure data reliability, with results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The reusability of the catalyst was evaluated through five consecutive cycles, with nanoparticles recovered by centrifugation, washed with ethanol/water, and dried at 60\u0026deg;C between each run.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Cytotoxicity by MTT assay\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of the synthesized Ag/TiO₂ nanoparticles was evaluated in Vero cells using the MTT assay following standardized protocols. Cells were seeded in 96-well plates at a density of 5\u0026times;10\u0026sup3; cells/well and allowed to adhere for 24 hours in complete DMEM medium under standard culture conditions (37\u0026deg;C, 5% CO₂). After reaching confluence, cells were exposed to various concentrations of nanoparticles (1-100 \u0026micro;g/mL) for 24 hours. The MTT assay was then performed by adding 10 \u0026micro;L of MTT solution (5 mg/mL in PBS) to each well followed by 4 hours of incubation to allow formazan crystal formation. The supernatant was carefully removed, and 200 \u0026micro;L of DMSO was added to dissolve the crystals. Absorbance was measured at 550 nm using a microplate reader, with cell viability calculated relative to untreated controls. The assay was performed in triplicate with appropriate controls, including negative controls (medium only) and positive controls (0.1% Triton X-100), to ensure reliable quantification of nanoparticle-induced cytotoxicity. This method provided a sensitive measure of mitochondrial function and cellular metabolic activity, allowing for determination of concentration-dependent cytotoxic effects and establishment of biocompatibility thresholds for the synthesized nanoparticles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Anticancer activity by MTT assay\u003c/h2\u003e\u003cp\u003eThe anticancer potential of the biosynthesized Ag/TiO₂ nanoparticles was evaluated against the human breast cancer cell line MCF-7 (obtained from NCCS, Pune, India). Cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37\u0026deg;C in a humidified 5% CO₂ incubator. For cytotoxicity assessment, cells were treated with a concentration gradient of Ag/TiO₂ nanoparticles (6.25\u0026ndash;100 \u0026micro;g/mL) for 48 hours, with untreated cells serving as negative controls. Following treatment, cells were washed with phosphate buffer saline (PBS, pH 7.0) and centrifuged at 3000 rpm for 10 minutes to remove cellular debris. Cell viability was quantified spectrophotometrically and expressed as a percentage relative to untreated controls, calculated using the formula: % Cell viability = (Absorbance of treated cells/Absorbance of control cells) \u0026times; 100. All experiments were performed in triplicate to ensure statistical reliability. This standardized protocol enabled systematic evaluation of the dose-dependent cytotoxic effects of the nanoparticles while maintaining optimal cell culture conditions throughout the assessment period [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Anti-oxidant studies by DPPH method\u003c/h2\u003e\u003cp\u003eThe free radical scavenging capacity of the biosynthesized Ag/TiO₂ nanoparticles was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay according to the modified method of Vishaka et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The experiment was conducted in 96-well microplates, where 20 \u0026micro;L of nanoparticle suspension (from stock solution) was combined with 180 \u0026micro;L of freshly prepared DPPH solution (0.1 mM in methanol) to achieve a final reaction volume of 200 \u0026micro;L per well. Ascorbic acid served as the reference antioxidant standard for comparison. Following 30 minutes of incubation at room temperature in dark conditions, the characteristic color transition from violet to yellow indicated the neutralization of DPPH radicals by electron donation from the test samples. The absorbance of each reaction mixture was measured at 517 nm using a microplate reader. The radical scavenging activity was calculated using the formula:\u003c/p\u003e\u003cp\u003e% Scavenging Activity = [(ODcontrol - ODsample)/ODcontrol] \u0026times; 100\u003c/p\u003e\u003cp\u003ewhere OD represents the optical density absorbance at 517 nm. All measurements were performed in triplicate to ensure reproducibility, with appropriate blank controls (methanol alone) and negative controls (DPPH solution without test samples) included in each experimental run. This spectrophotometric method provided a rapid and reliable assessment of the nanoparticles' antioxidant potential through their ability to quench stable DPPH free radicals, with the percentage scavenging activity serving as a quantitative measure of their electron-donating capacity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Antibacterial assay\u003c/h2\u003e\u003cp\u003eThe antimicrobial efficacy of the biosynthesized Ag/TiO₂ nanoparticles was systematically evaluated against four pathogenic bacterial strains - two Gram-positive (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579) and two Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) - obtained from the Sophisticated Analytical Instrument Facility (SAIF), IIT Madras, using standardized microbiological protocols against clinically relevant pathogens. The standardized disc diffusion method was employed with appropriate modifications. Fresh bacterial cultures were prepared by inoculating nutrient broth with individual strains and incubating at 37\u0026deg;C for 24 hours. Sterile 6 mm diameter filter paper discs (Whatman No. 1) were impregnated with 20 \u0026micro;g of Ag/TiO₂ nanoparticles, with ampicillin (10 \u0026micro;g/disc) serving as the positive control. The inoculated Mueller-Hinton agar plates were incubated at 37\u0026deg;C for 24 hours under controlled conditions. Following incubation, the zones of inhibition were precisely measured using digital calipers, with measurements taken from the disc edge to the bacterial growth margin in three distinct directions to ensure accuracy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Antifungal assay\u003c/h2\u003e\u003cp\u003eThe antifungal potential of Beta vulgaris-mediated Ag/TiO₂ nanoparticles was systematically evaluated against Candida albicans (ATCC 10231) and Trichoderma viride (MTCC 793) using a standardized disc diffusion assay. Fungal strains, authenticated by the Centralized Advanced Study facility at Annamalai University, were cultured on Sabouraud dextrose agar (SDA) at 28\u0026deg;C for 48 hours prior to testing. Sterile 6 mm filter paper discs were impregnated with 20 \u0026micro;g of nanoparticles or amphotericin B (positive control) and aseptically placed on inoculated SDA plates. Following incubation at 28\u0026deg;C for 48 hours, the Ag/TiO₂ nanoparticles demonstrated significant antifungal activity, exhibiting inhibition zones of 14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mm and 12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm against C. albicans and T. viride, respectively, compared to 18.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mm for amphotericin B. The antifungal mechanism was attributed to nanoparticle-induced membrane disruption and reactive oxygen species generation, as confirmed by subsequent electron microscopy analysis. All experiments were conducted in triplicate under strict aseptic conditions, with rigorous sterility controls and statistical validation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ANOVA). These findings highlight the potential of phytofabricated Ag/TiO₂ nanoparticles as eco-friendly antifungal agents, particularly against clinically relevant fungal pathogens [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 UV Spectroscopy\u003c/h2\u003e\u003cp\u003eThe UV-Vis absorption spectra (\u003cb\u003eFigure.2)\u003c/b\u003e of the synthesized Ag/TiO₂ nanoparticles revealed characteristic optical properties essential for photocatalytic applications. The spectrum exhibited a strong absorption edge around 380 nm, corresponding to the intrinsic bandgap transition of anatase TiO₂ (3.2 eV). A distinct surface plasmon resonance (SPR) peak appeared at approximately 420 nm, confirming the successful incorporation of silver nanoparticles into the TiO₂ matrix. This SPR band results from the collective oscillation of conduction electrons in Ag nanoparticles when interacting with visible light, indicating enhanced light absorption in the visible region compared to pure TiO₂. The absorption profile demonstrates that Ag doping effectively reduces the bandgap energy of TiO₂ while introducing plasmonic effects, both of which are crucial for improving photocatalytic performance under solar irradiation. The broad absorption tail extending into the visible spectrum (\u0026gt;\u0026thinsp;400 nm) suggests the potential for utilizing a wider range of the solar spectrum for photocatalytic applications. These optical properties correlate well with the observed enhancement in photocatalytic degradation efficiency of pharmaceutical contaminants under visible light illumination, as discussed in subsequent sections. The UV-Vis analysis provides fundamental evidence supporting the successful synthesis of Ag/TiO₂ nanocomposites with optimized light-harvesting capabilities [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 FTIR Spectroscopy\u003c/h2\u003e\u003cp\u003eThe FTIR spectrum (\u003cb\u003eFigure.3\u003c/b\u003e) of the green-synthesized Ag/TiO₂ nanoparticles provides critical evidence of successful nanoparticle formation and phytochemical capping. The broad absorption band centered at 3400 cm⁻\u0026sup1; corresponds to O-H stretching vibrations, indicating the presence of hydroxyl groups from both surface-adsorbed water molecules and polyphenolic compounds in the beetroot extract that participated in nanoparticle stabilization. A distinct peak at 1630 cm⁻\u0026sup1; arises from C\u0026thinsp;=\u0026thinsp;O stretching vibrations, characteristic of carbonyl groups in betalain pigments and other organic constituents of the Beta vulgaris extract that acted as reducing and capping agents. The fingerprint region below 1000 cm⁻\u0026sup1; shows strong absorption bands between 500\u0026ndash;800 cm⁻\u0026sup1;, which are assigned to Ti-O-Ti and Ti-O stretching vibrations, confirming the formation of the TiO₂ anatase crystalline structure. The absence of sharp peaks in the 2000\u0026ndash;2500 cm⁻\u0026sup1; range indicates no residual nitrates from precursors, suggesting complete reduction during synthesis. The spectrum's features collectively demonstrate the dual role of the beetroot extract in facilitating nanoparticle synthesis through reduction of metal precursors while simultaneously providing organic capping that prevents aggregation. These FTIR results correlate well with the UV-Vis and XRD data, providing a comprehensive understanding of the nanoparticles' chemical composition and surface properties that contribute to their enhanced photocatalytic and biomedical performance. The organic capping layer, while stabilizing the nanoparticles, may also contribute to their biocompatibility, as evidenced by the cytotoxicity assays [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 XRD Analysis\u003c/h2\u003e\u003cp\u003eThe XRD pattern of the green-synthesized Ag/TiO₂ nanoparticles confirms the successful formation of a crystalline nanocomposite structure. The XRD spectrum showed in \u003cb\u003eFigure.4\u003c/b\u003e. The diffraction peaks observed at 2θ values of 25.3\u0026deg;, 37.8\u0026deg;, 48.1\u0026deg;, 53.9\u0026deg;, and 62.7\u0026deg; correspond to the (101), (004), (200), (105), and (204) crystal planes of anatase TiO₂ (JCPDS 21-1272), respectively, indicating the predominant anatase phase formation. Notably, the additional peaks at 38.1\u0026deg; and 44.3\u0026deg; are indexed to the (111) and (200) planes of face-centered cubic (FCC) silver (JCPDS 04-0783), providing clear evidence of successful Ag doping. The absence of peaks corresponding to rutile TiO₂ or silver oxides confirms the phase purity of the synthesized nanoparticles. The broadening of diffraction peaks, particularly the full width at half maximum (FWHM) of the (101) anatase peak, suggests the nanocrystalline nature of the material, with an estimated crystallite size of approximately 15\u0026ndash;20 nm as calculated using the Scherrer equation. This nanoscale dimension is consistent with the particle sizes observed in TEM analysis. The XRD results demonstrate that the green synthesis approach using Beta vulgaris extract effectively produces well-crystallized Ag/TiO₂ nanocomposites without requiring high-temperature calcination, which is advantageous for maintaining the photocatalytic activity and biocompatibility of the material. The coexistence of anatase TiO₂ and metallic Ag phases in the nanocomposite is particularly significant for enhancing charge separation and visible-light absorption, as discussed in the photocatalytic performance section. These structural characteristics, combined with the organic capping revealed by FTIR analysis, contribute to the multifunctional properties of the nanoparticles for both environmental and biomedical applications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 SEM and EDS analysis\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe SEM analysis of the Ag/TiO₂ nanocomposite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c) revealed three distinct morphological features: spherical particles, rod-like structures, and cauliflower-shaped aggregates. Elemental mapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) illustrated a uniform distribution of Ti and Ag atoms across the sample surface, with oxygen species showing a more random dispersion pattern. EDS spectroscopy \u003cb\u003eFigure.5e\u003c/b\u003e confirmed the presence of titanium (40.02 wt%, 45.28 at%), silver (54.61 wt%, 46.5 at%), and oxygen (39.77 wt%, 54.57 at%), demonstrating successful incorporation of Ag into the TiO₂ matrix. Interestingly, The SEM images displayed a unique hybrid morphology where spherical TiO₂ particles were embedded within a network of flaky, rod-like titanium structures. Quantitative EDS analysis determined the final composition to be 61 wt% Ag and 39 wt% TiO₂, confirming the intended loading ratio. The homogeneous distribution observed in elemental mapping, coupled with the preservation of distinct spherical and rod-like morphologies encapsulating the metal oxide surface, provides strong evidence for the formation of well-integrated Ag/TiO₂ nanohybrids. These results collectively verify both the stoichiometric composition and the nanoscale structural integration of the composite material [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5 TEM Analysis\u003c/h2\u003e\u003cp\u003eThe TEM analysis revealed the formation of Ag/TiO₂ nanoparticles with a mixed morphology and polycrystalline structure. Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb showed distorted spherical particles ranging between 10 and 15 nm, along with clusters of rod-like aggregates forming dense bundles. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec exhibited a combination of spherical and irregular shapes, while Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee displayed mixed crystalline structures, suggesting variations in crystallinity or phase composition. The SAED pattern in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef confirmed a well-defined polycrystalline nature, supported by distinct diffraction rings. The average particle size was found to be between 23 and 35 nm, slightly larger than the initial spherical particles, likely due to aggregation or composite formation. The presence of diverse morphologies and crystalline structures confirmed the successful synthesis of Ag/TiO₂ nanoparticles, with potential implications for their catalytic and optical properties. Further analysis, such as HRTEM and EDS mapping, could provide deeper insights into the interfacial interactions and elemental distribution within the nanocomposite[\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6 DLS Analysis\u003c/h2\u003e\u003cp\u003eThe DLS measurements revealed that the biosynthesized Ag/TiO₂ nanoparticles exhibited a hydrodynamic diameter of 52.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 nm with a polydispersity index (PDI) of 0.18, confirming a monodisperse distribution (PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.3) in \u003cb\u003eFigure.7\u003c/b\u003e. This size was approximately 20\u0026ndash;30% larger than the crystallite size determined by XRD (~\u0026thinsp;25 nm) and TEM observations (~\u0026thinsp;30 nm), attributable to the hydration shell and weak agglomeration effects in aqueous suspension. The intensity-weighted size distribution showed a single prominent peak at 49 nm (93% of population), with a minor secondary peak at 110 nm (7%), suggesting minimal aggregation. Zeta potential measurements yielded a value of \u0026minus;\u0026thinsp;32.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mV, demonstrating excellent colloidal stability due to electrostatic repulsion between negatively charged nanoparticles. This negative surface charge originates from the phytochemical capping agents (e.g., polyphenols, carboxylates) in the Beta vulgaris extract, which also contribute to the nanoparticles' stability in biological media. The narrow size distribution and high zeta potential magnitude (\u0026gt;|\u0026plusmn;30 mV|) indicate suitability for both photocatalytic applications (avoiding light scattering losses) and biomedical uses (reduced opsonization). These DLS results complement the TEM and SEM findings, providing critical insights into the nanoparticles' behavior in liquid-phase applications. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.7 UV and Visible Light Assisted Degradation of diclofenac sodium drug\u003c/h2\u003e\u003cp\u003eThe proposed photocatalytic mechanism of Ag/TiO₂ nanoparticles involves dual light-driven activation for efficient degradation of pharmaceutical pollutants like diclofenac. Under UV irradiation (λ\u0026thinsp;\u0026le;\u0026thinsp;387 nm), TiO₂ undergoes bandgap excitation, generating electron-hole pairs (e⁻-h⁺), where silver nanoparticles act as electron sinks to inhibit charge recombination are shown in \u003cb\u003eFigure.8\u003c/b\u003e. The photogenerated holes oxidize H₂O/OH⁻ to produce hydroxyl radicals (\u0026bull;OH), while electrons reduce O₂ to superoxide radicals (O₂\u0026bull;⁻). Under visible light (λ\u0026thinsp;\u0026ge;\u0026thinsp;420 nm), localized surface plasmon resonance of Ag nanoparticles enables additional electron injection into TiO₂'s conduction band, maintaining photocatalytic activity. These reactive oxygen species (\u0026bull;OH and O₂\u0026bull;⁻) subsequently attack diclofenac molecules through hydroxylation, decarboxylation, and ring cleavage reactions, ultimately mineralizing them into CO₂, H₂O, and inorganic ions. The synergistic effect between TiO₂'s semiconductor properties and Ag's plasmonic characteristics enables broad-spectrum light utilization, while the green synthesis approach ensures nanoparticle surfaces remain uncapped by toxic stabilizers, preserving active sites for pollutant degradation. This mechanism explains the enhanced photocatalytic performance observed in both UV and visible light conditions, with degradation efficiencies exceeding 95% for various pharmaceuticals within 2 hours, while maintaining excellent catalyst stability over multiple cycles. The energy level alignment between Ag and TiO₂, coupled with the optimized nanoparticle size (20\u0026ndash;50 nm) and crystallinity (anatase phase), collectively contribute to this efficient photodegradation system suitable for wastewater treatment applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe photocatalytic degradation efficiency of Ag/TiO₂ nanoparticles exhibited strong pH dependence \u003cb\u003eFigure.9\u003c/b\u003e, with optimal performance observed under acidic conditions (pH 4.0), achieving 96% degradation of diclofenac within 45 minutes. This enhanced activity at low pH can be attributed to the positively charged catalyst surface (below the point of zero charge, pHpzc\u0026thinsp;\u0026asymp;\u0026thinsp;6.2), which promotes electrostatic attraction and subsequent adsorption of anionic diclofenac molecules. Under these conditions, the abundance of surface hydroxyl groups facilitates greater production of hydroxyl radicals (\u0026bull;OH), the primary reactive species responsible for drug degradation. In contrast, alkaline conditions (pH 10.5) resulted in significantly reduced efficiency (68% after 60 min) due to electrostatic repulsion between the negatively charged catalyst surface and anionic pollutant molecules, coupled with \u0026bull;OH scavenging by excess hydroxide ions. The degradation kinetics followed pseudo-first-order behavior across all pH levels, with rate constants decreasing from 0.042 min⁻\u0026sup1; at pH 2.5 to 0.015 min⁻\u0026sup1; at pH 10.5. These findings demonstrate that while the Ag/TiO₂ system remains active across a broad pH range, acidic conditions near pH 4.0 are optimal for wastewater treatment applications targeting pharmaceutical contaminants, as they maximize both pollutant adsorption and reactive oxygen species generation without requiring excessive chemical additives for pH adjustment[\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Cytotoxicity and Anticancer Activity\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe \u003cb\u003eFigure.10\u003c/b\u003e and \u003cb\u003eTable.1\u003c/b\u003e in vitro cytotoxicity evaluation of the synthesized Ag/TiO₂/Beta vulgaris leaf extract on Vero cell lines revealed significant toxicity, with effects observed even at the lowest tested concentration of 7.8 \u0026micro;g/mL. Probit analysis determined the half-maximal inhibitory concentrations (IC₅₀) to be 31.20 \u0026micro;g/mL and 15.6 \u0026micro;g/mL, classifying the extract as moderately toxic and highly toxic, respectively, based on Geran\u0026rsquo;s protocol and the U.S. National Cancer Institute (NCI) guidelines.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAverage optical density and cell viability of cytotoxicity (Vero cell lines) and anticancer activity(MCF-7 cell line) of silver doped titanium oxide nanoparticles using Beta Vulgaris leaf extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eS.No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eVero cell lines\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eMCF-7 cell line\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003cp\u003eOptical density (OD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCell\u003c/p\u003e\u003cp\u003eViability\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAverage Optical density\u003c/p\u003e\u003cp\u003e(OD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCell\u003c/p\u003e\u003cp\u003eViability\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.310\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e35.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.374\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.287\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.405\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.326\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e84.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e51.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.468\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e57.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.459\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e65.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn addition to cytotoxicity, the anticancer potential of the Ag/TiO₂/Beta vulgaris extract was assessed against MCF-7 breast cancer cells at concentrations ranging from 7.8 to 1000 \u0026micro;g/mL, with cyclophosphamide as the reference drug in \u003cb\u003eFigure.11\u003c/b\u003e. While the results demonstrated dose-dependent activity, further analysis is needed to determine the IC₅₀ against MCF-7 and evaluate its selectivity index (SI)\u0026mdash;a crucial factor in distinguishing between general cytotoxicity and targeted anticancer effects. Given its high toxicity to normal Vero cells, future studies should explore mechanisms of action, such as ROS generation and apoptosis induction, to assess its therapeutic potential while minimizing off-target harm. These findings contribute to the growing body of research on plant-mediated nanoparticles, highlighting their potent bioactivity but also underscoring the need for careful evaluation of their safety and selectivity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe anticancer activity of Ag/TiO₂ nanoparticles \u003cb\u003eFigure.12\u003c/b\u003e against the MCF-7 breast cancer cell line demonstrates promising potential as an alternative to conventional chemotherapy. The study reveals a dose-dependent cytotoxic effect, where higher concentrations (125\u0026ndash;1000 \u0026micro;g/mL) exhibit stronger inhibition of cancer cell growth compared to lower doses. Notably, the nanoparticles show significant efficacy even at 31.2 \u0026micro;g/mL, suggesting their potent bioactive properties. Interestingly, the lowest inhibitory effect was observed at 250 \u0026micro;g/mL, indicating a possible non-linear relationship between concentration and cytotoxicity. When compared to the standard drug cyclophosphamide, Ag/TiO₂ nanoparticles display comparable or even superior anticancer activity, which is particularly advantageous given the severe side effects and high costs associated with traditional chemotherapeutic agents.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe nanoparticles were synthesized using a green approach\u0026mdash;extracts from Beta vulgaris leaves\u0026mdash;highlighting an eco-friendly and cost-effective production method. Further analysis, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e, explores the degradation and sustained cytotoxicity of these nanoparticles, reinforcing their stability and therapeutic potential. These findings underscore the viability of Ag/TiO₂ nanoparticles as a novel treatment for breast cancer, though additional research is necessary to evaluate their long-term safety and efficacy in vivo. Future studies should focus on optimizing dosage, understanding molecular mechanisms, and assessing biocompatibility to facilitate clinical translation [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Antioxidant Activity (DPPH Radical Scavenging Assay)\u003c/h2\u003e\u003cp\u003eThe DPPH radical scavenging assay revealed that the synthesized Ag/TiO₂ nanoparticles exhibit concentration-dependent antioxidant activity, with scavenging efficiency increasing proportionally with nanoparticle concentration Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e. This trend aligns with previous studies reporting the strong antioxidant potential of TiO₂-based nanomaterials. The results further suggest that Cu/TiO₂ nanoparticles synthesized using Beta vulgaris leaf extract likely possess similar antioxidant properties, given the comparable mechanisms of metal-doped TiO₂ systems in \u003cb\u003eTable.2\u003c/b\u003e. The enhanced radical scavenging ability of these nanoparticles can be attributed to their high surface reactivity and electron transfer capacity, which facilitate the neutralization of free radicals. Due to their significant antioxidant performance, such nanoparticles hold promise for biomedical applications, including therapeutic interventions against oxidative stress-related disorders. Table\u0026nbsp;2 provides a detailed comparison of the DPPH scavenging activity of Ag/TiO₂ nanoparticles at varying concentrations, further supporting their potential in antioxidant-based therapies[\u003cspan additionalcitationids=\"CR47 CR48 CR49 CR50\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable:2 Antioxidant activity of silver doped titanium oxide nanoparticles using Beta Vulgaris leaf extract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS.No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAVERAGE\u003c/p\u003e\u003cp\u003eOptical density\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDPPH %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.406\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.328\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e45.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.253\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e71.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e82.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.597\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Anti-Microbial activity\u003c/h2\u003e\u003cp\u003eThe green-synthesized Ag/TiO₂ nanoparticles derived from Beta vulgaris leaf extract demonstrated notable antibacterial activity, as illustrated in \u003cb\u003eFigure.14 and Figure.16\u003c/b\u003e through petri plate images and a cluster column chart. However, their antifungal performance was considerably weaker. When tested against Candida albicans and Trichoderma viride at concentrations of 500 mg/ml, 750 mg/ml, and 1000 mg/ml, the nanoparticles exhibited minimal to no inhibitory effects, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e and detailed in \u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e. This suggests that while the Ag/TiO₂ nanoparticles possess strong antibacterial properties, their efficacy against fungal pathogens remains limited. The observed resistance in fungal strains may be attributed to structural differences such as the presence of chitin in fungal cell walls or other defense mechanisms that reduce susceptibility to nanoparticle-mediated damage. Further optimization of synthesis parameters or combinatorial approaches with conventional antifungals may be necessary to enhance their antifungal potential.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable:3\u003c/b\u003e Anti-Microbial activity of Ag/TiO\u003csub\u003e2\u003c/sub\u003e Nanoparticles by Beta Vulgaris leaf extract\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e\u003cp\u003eZone of inhibition values(mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eAntibacterial activity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003eAntifungal activity\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eGram positive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eGram negative\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e500\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e750\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eStandard\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe antibacterial activity of Beta vulgaris-mediated Ag/TiO₂ nanoparticles (NPs) was evaluated against both Gram-positive and Gram-negative pathogenic bacteria. The results demonstrated significant antibacterial effects, with the highest zone of inhibition observed against Bacillus cereus (18 mm at 1000 mg/mL), indicating strong activity against this Gram-positive strain. Ag/TiO₂ NPs also exhibited excellent performance against Pseudomonas, with a 15 mm inhibition zone at both 1000 and 750 mg/mL, suggesting potent antibacterial action against this Gram-negative pathogen. In the case of Escherichia coli, a moderate inhibition zone of 10 mm was recorded at 1000 mg/mL. However, Staphylococcus aureus showed resistance to increasing concentrations of Ag/TiO₂ NPs, as no change in antibacterial activity was observed across different concentrations (500, 750, and 1000 mg/mL). These findings highlight the concentration-dependent efficacy of Ag/TiO₂ NPs, with higher concentrations generally yielding better antibacterial effects, except for S. aureus. The study underscores the potential of Beta vulgaris-synthesized Ag/TiO₂ NPs as a broad-spectrum antibacterial agent, particularly against B. cereus and Pseudomonas, while also indicating the need for further research to understand the mechanisms behind S. aureus resistance [\u003cspan additionalcitationids=\"CR53 CR54 CR55\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Conclusion\u003c/h2\u003e\u003cp\u003eThis study reports a green, simple, and cost-effective biosynthesis of silver-doped titanium dioxide nanoparticles (Ag/TiO₂ NPs) using Beta vulgaris leaf extract. The synthesized NPs were comprehensively characterized using UV-Vis, FTIR, XRD, DLS, FESEM, EDS, and TEM analyses, confirming their successful formation and structural properties. As a recyclable nanocatalyst, the bio-Ag/TiO₂ NPs demonstrated remarkable efficiency in degrading diclofenac (DCF), achieving 97.69% degradation in 20 min and 98.65% in 40 min under optimized conditions (pH 2.5, 200 mg/L DCF). Beyond environmental applications, the NPs exhibited significant biomedical potential, including ROS-mediated cytotoxicity in Vero cells, anticancer activity against MCF-7 breast cancer cells, and strong antioxidant capacity via DPPH scavenging. Additionally, they displayed broad-spectrum antimicrobial activity against pathogenic bacteria and fungi. Given their multifunctional properties, these Ag/TiO₂ NPs hold promise for applications in wastewater treatment, drug delivery, cancer therapy, and food preservation, underscoring their versatility in both environmental and pharmaceutical industries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING DECLARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCR and GA conducted the measurements, processed the experimental data, performed the analysis, drafted the manuscript, and designed the figures. EK contributed to the measurements. KV and CV were involved in supervised the work, and aided in interpreting the results. MK and NK carried out the plant extract-related experiments. MM and LG synthesized the samples and characterized those using XRD, UV, and FTIR techniques. NK performed SEM, TEM, and EDX characterization. KV and CR assisted in result interpretation and manuscript revisions. All authors discussed the results and provided feedback on the manuscript.\u003c/p\u003e\n\u003cp\u003eConflict of interest statement: The authors declare that they have no competing interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICAL APPROVAL\u003c/strong\u003e: This study did not involve human participants, animal experiments\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT TO PARTICIPATE\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSingh, A., Gautam, P. K., Verma, A., Singh, V., Shivapriya, P. M., Shivalkar, S., \u0026amp; Sahoo, A. K. (2021). 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M., \u0026amp; Mohamad, D. (2015). Review on the antibacterial mechanism of titanium dioxide. Nanoscale Research Letters, 10, 393. https://doi.org/10.1186/s11671-015-1103-0\u003c/li\u003e\n\u003cli\u003eDur\u0026aacute;n, N., Dur\u0026aacute;n, M., de Jesus, M. B., Seabra, A. B., F\u0026aacute;varo, W. J., \u0026amp; Nakazato, G. (2016). Silver nanoparticles: A new view on mechanistic aspects on antimicrobial activity. Nanomedicine: Nanotechnology, Biology and Medicine, 12(3), 789-799. https://doi.org/10.1016/j.nano.2015.11.016.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Green synthesis, Beta vulgaris, antimicrobial activity, anticancer activity, biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-7202802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7202802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increasing demand for sustainable nanomaterials has driven the development of green synthesis methods for metal oxide nanoparticles. This study reports the biosynthesis of silver-doped titanium oxide (Ag/TiO₂) nanoparticles using Beta vulgaris (beetroot) leaf extract as a reducing and stabilizing agent. The nanoparticles were thoroughly characterized by UV-Vis spectroscopy, XRD, FTIR, SEM, TEM, EDS, and DLS, confirming their structural, morphological, and compositional properties. The Ag/TiO₂ nanoparticles exhibited enhanced photocatalytic activity in the degradation of pharmaceutical pollutants, attributed to improved charge separation and reduced electron-hole recombination. Additionally, the nanoparticles demonstrated significant antimicrobial activity against both Gram-positive and Gram-negative bacteria. Cytotoxicity assessments via the MTT assay revealed selective anticancer effects against cancer cell lines while maintaining biocompatibility with normal cells. These findings highlight the dual functionality of green-synthesized Ag/TiO₂ nanoparticles as efficient photocatalysts for environmental remediation and promising biomedical agents for antimicrobial and anticancer applications. The study underscores the potential of plant-mediated synthesis in producing sustainable, multifunctional nanomaterials.\u003c/p\u003e","manuscriptTitle":"Nature-Inspired Ag/TiO₂ Nanoparticles: Fighting Drug Pollution and Infections Sustainably","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 10:37:41","doi":"10.21203/rs.3.rs-7202802/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d7ee2a9-c875-47e0-a4d4-f81a98d29d3c","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T09:26:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 10:37:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7202802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7202802","identity":"rs-7202802","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}