Zinc Oxide and Titanium Dioxide Nanoparticles Bio fabricated for Enhanced Antimicrobic, Antioxidant, and Antitumor Performance

Zinc Oxide and Titanium Dioxide Nanoparticles Bio fabricated for Enhanced Antimicrobic, Antioxidant, and Antitumor Performance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zinc Oxide and Titanium Dioxide Nanoparticles Bio fabricated for Enhanced Antimicrobic, Antioxidant, and Antitumor Performance Tayyab Shafiq, Humaira Yasmin, Li Duan, Nukhbat Ullah, Tariq Nadeem, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6961371/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2025 Read the published version in BioNanoScience → Version 1 posted 12 You are reading this latest preprint version Abstract Background: Green nanotechnology has led the development of novel materials to address the growing concerns of antimicrobial resistance and effective cancer therapies. In the current study, we investigated the potential of biogenic zinc oxide (CV-ZnO ) and titanium dioxide (CV-TiO 2 ) nanoparticles (NPs) synthesized using Cinnamomum verum bark extract to combat human pathogens and assess anticancer potential. Methods: Green synthesized CV-ZnO and CV-TiO 2 nanoparticles were characterized using UV-vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), and Scanning Electron Microscopy (SEM). Their antibacterial and antifungal properties were assessed against a panel of pathogenic bacteria and fungi using the disc diffusion method. The antioxidant, cytotoxic, and anti-inflammatory potential was examined against the Huh-7 liver cancer cell line. Results: The biogenic spherical-shaped CV-ZnO and CV-TiO 2 nanoparticles exhibited sizes ranging from 40 to 80 nm with absorption peaks at 300-320 nm and 300-400 nm, respectively. FTIR and XRD patterns indicated the presence of hydroxyl and organic groups, confirming high crystallinity, stabilization and phase purity. Both types of NPs exhibited significant antibacterial and antifungal activities, with larger zones of inhibition at higher concentrations. The CV-TiO 2 nanoparticles showed superior antioxidant activity and induced higher levels of superoxide dismutase in Huh-7 cells compared to CV-ZnO nanoparticles. Furthermore, these nanoparticles, especially CV-TiO 2 , exhibited potent cytotoxicity (67.67% at 100 μg/ml) against Huh-7 liver cancer cells in a dose-dependent manner, accompanied by the modulation of key apoptotic (Bax) and inflammatory genes (AFP, Bcl-2, PTEN). Conclusion: The current findings suggest the potential application of biogenic CV-ZnO and CV-TiO 2 NPs in developing novel antimicrobial agents and cancer therapies. Further translational studies are warranted to explore their clinical application. Green synthesis CV-ZnO nanoparticles CV-TiO2 nanoparticles Cinnamomum verum Antibacterial Antifungal Anticancer Nanomedicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Nanotechnology has embarked multidisciplinary applications in the field of research with profound developments. The synthesis of metal and metal oxide nanoparticles (NPs) involves the use of depleting and balancing agents to produce materials with distinct characteristics (Rastogi et al., 2017 ) (Agarwal et al., 2017 ). Plants are reported to be capable of reducing metal ions. Many factors influence the synthesis of nanoparticles, including pH, temperature, and reaction time (Tiguntseva et al., 2017 ) (Agrawal and Rathore, 2014 ). CV-ZnO has been categorized as a safe metal oxide by the FDA (Agrawal et al., 2017 ). It is used in a variety of consumer items, including lotions, ceramics, wastewater treatment, and rubber processing. CV-ZnO absorbs UV radiation and is consequently used in cosmetics. The antibacterial properties make it odour-resistant, and its anticancer properties render it a promising tumour therapy option (Agrawal et al., 2017 , Agarwal et al., 2017 ). Titanium dioxide (CV-TiO 2 ) nanoparticles have long been regarded as low-toxicity, weakly soluble particles. They are used as a negative control in particle toxicology research, both in vitro and in vivo . In recent years, CV-TiO 2 NPs have become increasingly popular in industrial and consumer applications due to their larger surface area per unit mass and catalytic activity. Recent studies suggest that CV-TiO 2 NPs exhibit different bioactivities (Shi et al., 2013 ). A variety of plants have been reported to benefit from titanium dioxide nanoparticles (CV-TiO 2 ), benefiting sustainable agriculture by reducing soil salinity. They are used as a foliar spray to boost plant growth enzyme activity, chlorophyll content photosynthesis, nutrient absorption, stress tolerance, yield, and crop quality. They poesses small size, easy handling, long-term storage, high efficacy, and nontoxicity (El-Said et al., 2014 ). Cinnamon is a potent spice that has been used medicinally for thousands of years. Cinnamon ranks first among twenty-six of the world's most popular herbs and medicinal spices due to its beneficial antioxidant levels. Cinnamon bark extract is a key bioactive component with a variety of biological activities, including antibacterial, antibiofilm, anthelmintic, anticancer, and antifungal properties (Ali et al., 2020 ). C. verum bark extract can be used to synthesize CV-ZnO and CV-TiO 2 NPs. In the current investigation, the biogenic one-step synthesis and capping of Zinc and titanium nanoparticles using cinnamon bark extract was carried out. Green synthesis of metallic nanoparticles has several advantages over chemical and physical production, including decreased toxicity, environmental friendliness, low energy consumption, and cost-effectiveness (Ali et al., 2020 ). Antimicrobial resistance is a growing global concern. Current medicinal practices and the widespread use of broad-spectrum antibiotics have exacerbated the raised concern. Similarly, antifungal resistance poses significant challenges, particularly for patients with invasive fungal infections affecting critical organs. Addressing these issues necessitates innovative approaches. Furthermore, the cytotoxic effects of metallic nanoparticles against cancer cell lines highlight their potential in cancer treatment. Due to their distinct physicochemical characteristics and versatile applications, nanoparticles are potential drug-delivery vehicles to treat cancer. (Lee et al., 2006). In the current investigation, we explored the promising potential of green synthesized bio-fabricated zinc oxide (CV-ZnO ) and titanium oxide (CV-TiO 2 ) nanoparticles as multifunctional materials with significant implications in combating human pathogens and exhibiting anti-cancerous activities. The synthesized nano biocomposites were characterized using various techniques, including XRD, FTIR, SEM and UV spectroscopy. Their antibacterial potential was assessed against four pathogenic bacterial strains, including Bacillus cereus, Staphylococcus aureus Enterobacter aerogenes and Escherichia coli. The antifungal potnential was evaluated against Alternaria solani, Macrophomina phaseolina, Aspergillus niger, Candida albicans . In vitro anticancer activity of these nano biocomposites was examined against the Huh-7 liver cancer cell line. Material and Methods Synthesis of Nanoparticles and Protocol Optimization Bark samples of Cinnamomum verum were initially rinsed with distilled water thrice and allowed to dry overnight. Following this, 30 g of bark was ground into a fine powder, and 20 g of the powder was added to 100 ml of water and heated on a hot plate for approximately 30 minutes at 50-60°C until the solution turned dark brown. The extract was then filtered using Whatman no. 1 filter paper to remove impurities, frozen, and stored at 5°C for future use. For the synthesis of CV-ZnO and CV-TiO 2 nanoparticles, two salt solutions were prepared at different concentrations. A 0.1 M solution of zinc nitrate (Sigma Aldrich, Zn(NO3)2·6H2O, CAS Number 10196-18-6) was prepared by dissolving 14.873 g of zinc nitrate in 500 ml of distilled water. Similarly, a 1 M solution of titanium chloride (Sigma Aldrich, TiCl4, CAS No: 7550-45-0) was prepared by dissolving 18.9 ml of titanium chloride in 100 ml of distilled water. Both solutions were stirred for two minutes using a magnetic stirrer to ensure complete dissolution and stored in 500 ml beakers. To achieve optimal synthesis conditions for CV-TiO 2 and CV-ZnO NPs, various parameters were optimized, including the quantity of plant extract, stirring time, reaction temperature, pH of the solution, and salt stability. The reaction mixture was then centrifuged at 5000 rpm for 5 minutes to obtain the nanoparticles. The supernatant was discarded, and the pellet was retained. Characterization of CV-TiO2 and CV-ZnO NPs Characterization of the synthesized CV-TiO 2 and CV-ZnO nanoparticles was performed using various techniques to determine their structural, optical, and morphological properties. UV-visible spectrophotometry: This technique was used to investigate the optical properties of the synthesized nanoparticles. The absorption spectra of the CV-ZnO and CV-TiO 2 NPs were recorded over a wavelength range of 200-1000 nm using a UV-Vis spectrophotometer. The samples were prepared by dispersing the nanoparticles in distilled water and sonicating them for 30 minutes to ensure a uniform suspension. The absorbance was measured. Fourier Transform Infrared Spectroscopy: FTIR spectroscopy was used to identify the functional groups and capping agents present on the surface of the nanoparticles. The FTIR spectra of the CV-TiO 2 and CV-ZnO NPs were recorded. The samples were prepared by mixing the nanoparticles with potassium bromide (KBr) to form pellets. The spectra were obtained in the range of 4000-400 cm -1 . X-Ray Diffraction (XRD): This method was conducted to determine the crystalline structure, phase purity, and average crystalline size of the synthesized nanoparticles. The XRD patterns of the CV-TiO 2 and CV-ZnO NPs were obtained using an X-ray diffractometer equipped with Cu Kα radiation (λ = 1.5406 Å). The samples were prepared by placing the nanoparticles on a glass slide. The data were collected over a 2θ range of 10° to 80° with a step size of 0.02°. Scanning Electron Microscopy: This technique was used to examine the surface morphology, size distribution, and overall shape of the nanoparticles. The SEM images of the CV-TiO 2 and CV-ZnO NPs were captured using a scanning electron microscope. The samples were prepared by placing a drop of nanoparticle suspension onto a silicon wafer, followed by drying at room temperature. The dried samples were coated with a thin layer of gold to enhance conductivity before imaging. Biomedical Applications of CV-TiO 2 and CV-ZnO NPs The synthesized CV-TiO 2 and CV-ZnO NPs were evaluated for their antimicrobial, antifungal, and antioxidant activities. Antibacterial Activity: The antimicrobial ability of the NPs was determined against four pathogenic bacterial strains, including Bacillus cereus, Staphylococcus aureus Enterobacter aerogenes and Escherichia coli, using the disc diffusion method. To assess the antibacterial activity of CV-TiO 2 and CV-ZnO nanoparticles (NPs), seven different concentrations were prepared (5, 10, 15, 20, 30, 50, 100, and 150 ppm). The suspensions of CV-ZnO NPs in water were sonicated for 20-30 minutes at 60°C to ensure complete solubilization of the NPs. The same procedure was for CV-TiO 2 NPs but with DMSO. The discs were soaked for approximately 20 minutes with varying concentrations of NPs and dried. Autoclaved media was poured into Petri plates and allowed to solidify for about an hour. A 10 µL concentration of bacterial strains from LB media was added to each Petri plate. The culture was evenly spread across the plate using a spreader. This procedure was performed for each bacterial strain, with three replicates for each strain. The sealed plates were placed in an incubator at room temperature for 24-48 hours. The plates were examined for the bactericidal activity of the NPs. The zones of inhibition around the discs were measured to determine the antibacterial efficacy. Antifungal Activity: The antifungal activity of the NPs was tested against Alternaria solani , Macrophomina phaseolina , Aspergillus niger , and Candida albicans using the disc diffusion method. The procedure was similar to the antibacterial activity assay, with different nanoparticle concentrations tested for antifungal efficacy (30, 50, 100 and 150 ppm). Water was used as a control. DPPH Assay The antioxidant activity was assessed using the DPPH radical scavenging assay. Various concentrations of the nanoparticle samples were mixed with 0.4 mM DPPH in ethanol, and the reaction mixtures were incubated for 30 minutes at room temperature. Absorbance was measured at 517 nm, and the percentage of radical scavenging activity was calculated. Superoxide dismutase (SOD) Assay According to the manufacturer's instructions, the SOD assay kit was used to assess the SOD activity in Huh-7 cells treated with CV-ZnO and CV-TiO 2 nanoparticles. For each well, 20 μL of the supernatant from different experimental groups was added, followed by the addition of 200 μL of WST solution. This included blanks 1, 2, and 3 as reference points. Subsequently, 20 μL of the enzyme working solution was added to all wells containing the samples and blank 1. The plate was gently mixed and incubated at 37 °C for 20 minutes. After incubation, the absorbance was measured at 450 nm using a microtiter plate reader. To ensure reproducibility, all samples were analyzed in triplicate. Cytotoxicity Assessment Assay We investigated the cytotoxic potential of CV-ZnO and CV-TiO 2 nanoparticles synthesized in the current investigation against Huh-7 liver cancer cells. To assess the cytotoxicity, a total of 5 × 10 4 cells were seeded in 96-well flat-bottom plates containing DMEM supplemented with 5% FBS. Subsequently, various concentrations of CV-ZnO and CV-TiO 2 nanoparticles (5, 10, 25, 50, 100 μg/mL) were added to the wells. The cells were incubated for 48 hours at 37 °C in a 5% CO 2 incubator. After that, the cell monolayers were rinsed with serum-free media, and 100 μL of a 5 mg/mL MTT solution was added to each well. The plates were incubated for 4–5 hours to allow the MTT into formazan crystals by viable cells. Following this, the media was aspirated, and the formazan crystals were dissolved in 100 μL of 0.1% DMSO. The absorbance of the resulting solution was measured at 550 nm using a microplate reader (Multiskan GO, Thermo Scientific, USA). In parallel, control cells treated with PBS buffer instead of green synthesized nanoparticles were taken as a negative control. The experiment was performed in triplicate. The percentage of cell inhibition was calculated using the formula [(Ac–As)/(Ac)] *100, where Ac represents the absorbance of the control wells, and As represents the absorbance of the sample wells. qRT-PCR-based expression profiling of proinflammatory and apoptotic genes Huh-7 cells were incubated in six-well plates for a duration of 24 hours, followed by treatment with 50 and 100 µg/ml of CV-TiO 2 nanoparticles. The purpose of this treatment was to isolate total RNA from the cells. To extract the RNA, Trizol was utilized in accordance with the manufacturer's instructions. The samples were collected from both control cells and those treated with stress and CV-TiO 2 nanoparticles. The concentration of the extracted RNA was determined by measuring the absorbance at 260 nm using a Nanodrop spectrophotometer. After ethanol washing (1 mL), the RNA was dissolved in 50 µL of 0.1% Diethyl pyrocarbonate (DEPC) treated water and stored at −80 °C until further use. For reverse transcription, the RevertAidTM first-strand synthesis kit (Thermo Scientific, Cat No: K1622) was used. Following the manufacturer's instructions, 1 µg of RNA was reverse transcribed into cDNA. Forward and reverse primers (0.5 µM each) specific to the genes of interest (AFP, Bax, Bcl-2, and PTEN) (Wang et al., 2021) were used in the study. Template cDNA (2 μL) was added to a final reaction volume of 20 µL. Real-time PCR was performed using a StepOne Plus thermocycler (Applied Biosystems) and SYBR Green PCR Master Mix (Thermofisher, catalogue number K0221). All real-time PCR assays were run in triplicate. The data were reported as the mean of three independent experiments. The transcriptomic expression of the selected genes was normalized with the GAPDH gene used as an internal control. Statistical Analysis Each experimental assay was performed in triplicate. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc, La Jolla, CA). The significance level for determining differences between mean values was set at p < 0.05. Results Optimization of Green Synthesis of CV-TiO 2 and CV-ZnO NPs Figure 1a shows the effect of varying the quantity of plant extract on the synthesis of CV-TiO 2 and CV-ZnO NPs. Optimal synthesis was achieved using 20 ml of plant extract for both CV-TiO 2 and CV-ZnO NPs with optimal size and stability. Figure 1b presents the effect of different stirring times on nanoparticle synthesis. The optimal stirring time for CV-TiO 2 and CV-ZnO NPs was found to be 120 minutes. This duration allowed for adequate interaction between the extract and the metal salts. CV-TiO 2 NPs showed optimal synthesis at 40°C, while CV-ZnO NPs showed the best results at 70°C (Figure 1c). These temperatures facilitated the efficient conversion of metal salts to stable and uniformly sized NPs. CV-TiO 2 and CV-ZnO NPs showed optimal synthesis at a pH of approximately 7 (Figure 1d). The optimal pH level ensured the proper ionization of the bioactive compounds in the extract, enhancing their reducing capability. Both nanoparticles remained stable under all tested salt concentrations (Figure 1e). Characterization of CV-TiO 2 and CV-ZnO NPs UV-Vis Spectrophotometry Maximum absorbance peak was observed between 300-400 nm during the synthesis of CV-TiO 2 NPs and around 300-320 nm during the synthesis of CV-ZnO NPs (Figure 2a). These peaks reflect their respective characteristic band gap absorption of CV-TiO 2 and CV-ZnO NPs. Fourier Transforms Infra-Red Spectroscopy (FTIR): The FTIR spectrum of CV-TiO 2 NPs (Figure 2b) indicates that there are various stretches at different levels giving different peaks (3550-3200 cm -1 strong O-H stretching, 3000-2840 cm -1 medium C-H stretching, 2000-1650 cm -1 weak C-H bending, 1000-650 cm -1 strong C-C bending, and 900-700 strong C-H bending). In the case of CV-ZnO NPs, slightly different vibration peaks were observed, including 3700-3584 cm -1 medium O-H stretching, 2400-2000 cm -1 strong O=C=O stretching, and 2000-1650 cm -1 weak C-H bending (Figure 2c). These peaks confirm the presence of functional groups involved in the stabilization and capping of both NPs. X-Ray Diffraction The XRD pattern of CV-TiO 2 NPs reveals the distinct peaks at 25.3°, 37.8°, 48.0°, 54.1°, and 62.7°, corresponding to the anatase phase of CV-TiO 2 (Figure 2d). These peaks confirm the crystalline nature and phase purity of the synthesized CV-TiO 2 nanoparticles. The CV-ZnO nanoparticles exhibited peaks at 31.7°, 34.4°, 36.3°, 47.5°, and 56.6°, characteristic of the wurtzite hexagonal phase of CV-ZnO (Figure 2e). These peaks indicate the crystallinity and phase purity of the CV-ZnO nanoparticles. Scanning Electron Microscopy (SEM) The SEM images were obtained at 10 µm resolution. The synthesized NPs possessed uniformly dispersed spherical shapes and fused agglomerates. Their uniform dispersion and spherical morphology indicate a narrow size distribution. CV-TiO 2 NPs were seen uniformly dispersed with a size ranging from 40 nm to 80 nm (Figure 3a). The SEM image of CV-ZnO NPs sizes also ranged from 40 nm to 80 nm with similar uniform dispersion (Figure 3b). Antioxidant activity of CV-TiO 2 and CV-ZnO NPs DPPH Assay The potential of CV-TiO 2 and CV-ZnO NPs to scavenge free radicals was evaluated by using a DPPH assay. The results show a concentration-dependent increase in antioxidant activity of both nanoparticles (Figure 4). The scavenging activity of CV-ZnO NPs was slightly lower than that of CV-TiO 2 NPs at higher concentrations (Figure 4c). At 50 µg/ml, the CV-TiO 2 NPs showed a maximum activity of 90% (Figure 4a). In the case of CV-ZnO NPs, it was 50% activity at the same concentration (Figure 4b). SOD Assay Superoxide Dismutase (SOD) is responsible for the conversion of superoxide ions into less harmful byproducts. The activity of this enzyme was examined in Huh-7 cells treated with CV-ZnO and CV-TiO 2 nanoparticles. The SOD activity was found to be higher in the CV-TiO 2 -treated cells compared to the CV-ZnO -treated cells at 25, 50, and 100 μg/ml concentrations (Figure 5). The percent SOD activity in Huh-7 cells treated with CV-ZnO nanoparticles at these concentrations was 34.94%, 46.16%, and 49.02%, respectively. In contrast, the percent SOD activity in Huh-7 cells treated with CV-TiO 2 nanoparticles was 42.76%, 53.34%, and 59.73%, respectively. Antibacterial Activity of CV-TiO 2 , CV-ZnO NPs Antibacterial activity of CV-TiO 2 and CV-ZnO NPs was observed against pathogenic bacterial strains ( B. cereus, S. aureus, E. coli, E. aerogenes ) as shown in Figure 6. CV-TiO 2 NPs (200 ppm) showed 86% inhibitions against B. cereus, 80% inhibition against S. aureus, 74% against E. coli and 71% against E. aerogenes (Figure 6a) . Non-significant percent inhibition was observed at 5 ppm against these bacterial strains. Similarly, CV-ZnO NPs at 200 ppm showed 76% inhibition against B. cereus, 75% against S. aureus, 74% against E. coli and 71% against E. aerogenes (Figure 6b) . CV-ZnO NPs exhibited non-significant percent inhibition at 5 ppm concentration against these bacterial strains. Antibacterial activity of plant extract of cinnamon verum showed 36% inhibition against B. cereus, 34% inhibition against S. aureus, 24% against E. coli and 22% against E. aerogenes. The antibiotic (ciprofloxacin) showed 56% inhibition against B. cereus, 51% inhibition against S. aureus, 61% against E . coli and 61% against E. aerogenes . Antifungal Activity of CV-TiO 2 and CV-ZnO NPs CV-TiO 2 NPs at 200 ppm showed 28% inhibition against Alternaria solani , 26% inhibition against Aspergillus niger, 22% against Macrophomina phaseolina and 21% against Candida albicans . At 50 ppm concentration, CV-TiO 2 NPs showed no percent inhibition against any fungal strains (Figure 7a). CV-ZnO NPs at 200ppm showed 23% inhibition against Alternaria solani , 21% inhibition against Aspergillus niger, 20% against Macrophomina phacelia and 19% against Candida albicans . At 50 ppm concentration, they showed no percentage inhibition against any fungal strain (Figure 7b). Cytotoxicity Assessment of CV-TiO 2 and CV-ZnO NPs Figure 8 depicts the impact of different concentrations of CV-ZnO and CV-TiO 2 nanoparticles on the proliferation of Huh-7 cancer cells. Notably, increasing the concentration of nanoparticles led to a decrease in cancer cell viability. CV-TiO 2 nanoparticles displayed a higher cytotoxic response compared to CV-ZnO nanoparticles across all concentrations. At concentrations of 50 μg/ml and 100 μg/ml of CV-ZnO nanoparticles, the cell viability decreased to 55.3% and 43.68%, respectively. Conversely, Huh-7 cells treated with CV-TiO 2 nanoparticles exhibited a cell viability of 44.17% and 32.329% at the respective concentrations. Quantitative RT-PCR Analysis For Apoptotic And Antiapoptotic Markers CV-TiO 2 nanoparticles exhibited better radical scavenging activity, SOD activity and cytotoxicity than CV-ZnO . As a result, the quantitative RT-PCR analysis was performed for Huh-7 cells treated with CV-TiO 2 nanoparticles to assess the mRNA expression levels of inflammation and apoptosis-associated genes (Figure 9). Proinflammatory cytokines (AFP, Bcl-2, and PTEN) were significantly elevated in the stress groups after stimulation with cobalt chloride. Green synthesized CV-TiO 2 nanoparticles significantly lowered the levels of these gene expressions in a dose-dependent manner compared to the stress group (Figures 9a, 9c, and 9d). The mRNA levels of the apoptotic gene (Bax) in Huh-7 cells treated with CV-TiO 2 resulted in its upregulation (Figure 9b). Discussion Metallic nanoparticles exhibit potent antimicrobial, antifungal and anticancer potential (Zhu et al., 2019 , Zhu et al., 2020 ) (Kumari et al., 2021 ) (Baer et al., 2010 ). In this study, we synthesized CV-TiO2 and CV-ZnO NPs (Chung et al., 2015 ) with the bark extract of Cinnamon Verum . Plant phytochemicals, including alkaloids and flavonoids, play a significant role in reducing the metallic salts and the nanoparticles are precipitated at the end of the reaction as byproducts (Yadav and Agarwala, 2011 ). Based on the above data, it can be stated that the optimal volume of Cinnamomum verum bark extract to be used is 20 ml, while lesser volumes of the extract led to poor formation of nanoparticles. This is quite in tandem with (Nazir et al., 2021 ) that stated that 15-25ml of plant extract was required in order to get the right bioactive content that will help in reduction and stabilisation of CV-ZnO nanoparticles Likewise, (Ahmad et al., 2024 ) (Ragavendran et al., 2023 ) established that adequate stirring was attained in 30 minutes to enhance the interfacial area for the synthesis of CV-ZnO nanoparticles Stated (Rathore et al., 2023 ) showed that 45 minutes of stirring was appropriate to ensure the proper distribution and stability of the CV-TiO 2 nanoparticles. Secondly, the current investigation specified the reaction temperature of form CV-ZnO nanoparticles to be 70°C and that of CV-TiO 2 nanoparticles to be 40°C. This supports (Rai et al., 2023 ) who postulated that the synthesis of CV-ZnO nanoparticles was most favorable at around 60- 75oC, and (Singh et al., 2023 ) who noted that synthesis of CV-TiO2 nanoparticles preferred at 40–50oC. Pretreatment of the plant extract was carried out at a neutral pH because it was important to achieve the optimal ionization of some of the compounds in the plant extracts to improve their ability to reduce and stabilize the metal ions loading into the reaction; this is in agreement with (Boro et al., 2024 ) who postulated that neutral pH should be used in the synthesis process of CV-ZnO and CV-TiO 2 Finally, our work pointed out that nanoparticle stability in saline conditions could be crucial if the nanoparticles are to be used in a biomediocre setting. Following the same line, (Patel et al., 2022 ) concluded that CV-ZnO and CV-TiO 2 nanoparticles produced from plant extracts had great stability in saline conditions for physiological uses. The amount of plant extract used in the synthesis process is critical to achieve nanoparticles with desirable characteristics. Our study tested various quantities of C. verum bark extract. The optimal quantity (20 ml) provided sufficient bioactive compounds necessary for the reduction and stabilization of the metal ions (Fig. 1 a). Insufficient amounts of extract resulted in suboptimal nanoparticle formation. The stirring time during the synthesis process significantly affects the interaction between the metal ions and the bioactive compounds in the plant extract (Fig. 1 b). The reaction temperature is another crucial factor that influences the rate of reaction and the quality of the synthesized nanoparticles. The optimal temperature for ZnO NPs synthesis was found to be 70°C, while for TiO2 NPs, it was 40°C (Fig. 1 c). Maintaining a neutral pH ensured the proper ionization of the plant extract compounds, enhancing their reducing and stabilizing capabilities (Fig. 1 d). The stability of nanoparticles in saline environments is essential for their potential biomedical applications. This resilience makes them suitable for applications in physiological environments where salt concentrations vary (Fig. 1 e). Nanomaterials are characterized using a variety of techniques (Akbari et al., 2011) (Baer et al., 2010 ) (Kumar and Dixit, 2017 ). For CV-TiO 2 NPs, a characteristic absorbance peak shows the anatase phase of CV-TiO2 (Fig. 2 a). This peak is indicative of the band gap absorption. The sharp and distinct peaks in the UV-Vis spectra affirm the successful synthesis of both NPs. The FTIR spectrum of CV-TiO 2 and CV-ZnO NPs displayed characteristic peaks (Fig. 2 b and 2 c). This highlights the effective capping and stabilization of the nanoparticles by the bioactive compounds in the C. verum bark extract containing hydroxyl and organic groups (Xulu et al., 2022 ). The XRD pattern of CV-TiO 2 and CV-ZnO NPs revealed distinct peaks (Fig. 2 d and 2 e). These sharp peaks confirm the high crystallinity and phase purity of the CV-TiO 2 and the wurtzite hexagonal phase of CV-ZnO NPs. The SEM images of CV-TiO2 and CV-ZnO nanoparticles revealed their uniform morphology and narrow size distribution (Figs. 3 a and 3 b). The slight difference in antioxidant activity between CV-TiO 2 and CV-ZnO nanoparticles could be attributed to their chemical properties and their interaction with the bioactive compounds in the extract (Figs. 4 and 5 ). The findings show that these nanoparticles have a substantial capacity to donate electrons or hydrogen atoms to neutralize free radicals. The presence of phenolic compounds and flavonoids in the bark extract likely contributes to the enhanced antioxidant activity (Xie et al., 2011 ). This antioxidant activity is significant in cancer therapy. The slightly higher antioxidant activity induced by CV-TiO 2 nanoparticles compared to CV-ZnO nanoparticles may be due to differences in their catalytic properties and surface chemistry. CV-TiO 2 nanoparticles exhibit photocatalytic activities, which could contribute to their superior performance (Morones et al., 2005 ). CV-TiO2 CV-ZnO The antibacterial mechanism of CV-TiO 2 NPs is likely due to the generation of reactive oxygen species (ROS) upon exposure to light. The antibacterial action of CV-ZnO NPs is primarily attributed to the release of Zn 2+ ions, which disrupt bacterial cell membrane integrity, leading to oxidative stress and cell death (Xie et al., 2011 ). NPs may continuously discharge ions which can bind to the cell wall and cytoplasmic membrane due to electrostatic attraction and affinity for sulphur proteins. The attached ions might increase the permeability of the cytoplasmic membrane which causes the bacterial envelope to be disrupted. Because sulphur and phosphorus are key components of DNA, the interaction of ions with these elements can impair DNA replication and cell reproduction. Furthermore, ions can prevent protein synthesis by denaturing ribosomes in the cytoplasm (Morones et al., 2005 ). From the results obtained it can be deduced that the concentration of cytotoxicity in the NPs is in concordance with the findings of (Khan et al., 2018 ) which showed toxicity in HCC cells in relation to the concentration of NPs. (Akbari et al., 2011) also provide evidence of mitochondrial impairment involved in cell death since the NPs cause apoptosis of cancer cells through the impairment of caspase pathways. Furthermore, we have also found that green synthesized NPs modulate the cellular redox potential, similar to the recent study by (Agrawal et al., 2017 )that described effects of green NPs on cellular oxidative stress and apoptosis. These comparisons reinforce the significance of our observations within the framework of the nanoparticle-based therapeutic approaches to HCC treatment.The superior antioxidant activity of CV-TiO 2 nanoparticles suggests their potential in combating oxidative stress, a hallmark of HCC (Li et al., 2020 ). Inflammation is a crucial driver of tumorigenesis. Our findings revealed that CV-TiO 2 nanoparticles significantly downregulated the expression of the proinflammatory cytokines in huh-7 HCC cells (Fig. 9 ). Numerous studies have demonstrated that alpha-fetoprotein (AFP) suppresses the immune system and is also involved in the MDR process in liver cancer patients. Previous research has shown that when treated with green synthesized nanoparticles (Iqbal et al., 2021 ), these inflammatory markers are down-regulated in various human cancer cell lines. PTEN regulates the PI3K/AKT signaling pathway. PTEN loss or mutation can lead to aberrant activation of this pathway and contribute to HCC development (Chen et al., 2019 ) (Zhao et al., 2020 ). Bcl-2 inhibits apoptosis. Its expression is abnormally elevated in HCC tissues (Nakopoulou et al., 1999 ). Down-regulation of Bcl-2 in HCC cells can inhibit the proliferation and invasion of cancer cells (Zhang et al., 2021 ). Bax regulates programmed cell death (Guo et al., 2005 ). Several studies have shown that Bax expression is reduced in HCC cells, which may confer resistance to apoptosis (Li et al., 2010 ). The imbalance between pro-apoptotic and antiapoptotic proteins contributes to tumor cell survival and resistance to therapy. In our study, CV-TiO 2 nanoparticles upregulated the expression of the pro-apoptotic gene Bax in HCC cells. The in vitro nature of our study restricts the translation of our findings to an in vivo setting. Further studies are warranted to evaluate the efficacy and safety of CV-TiO 2 and CV-ZnO NPs in preclinical and clinical settings. Our study primarily focused on the cytotoxic effects and gene expression changes of green synthesized nanoparticles. It is essential to investigate nanoparticle biodistribution, cellular uptake, and long-term toxicity. Conclusion The current investigation demonstrated the successful synthesis and characterization of CV-TiO 2 and CV-ZnO nanoparticles using Cinnamomum verum bark extract. The characterization techniques, including UV-Vis spectrophotometry, FTIR spectroscopy, XRD analysis, and SEM imaging, confirmed the formation of uniformly dispersed, spherical nanoparticles with high crystallinity and phase purity. The synthesized nanoparticles exhibited significant antioxidant, antibacterial, and antifungal activities. These nanoparticles possessed potent cytotoxic and anti-inflammatory effects against Huh-7 liver cancer cells. Future studies should focus on in vivo evaluations, long-term safety assessments, and their possible mechanisms of action. The clinical translation of these nanoparticles into therapeutic formulations could pave the way for new and effective treatment regimens for infections and cancer. Declarations Ethics approval and consent to participate Not Applicable Consent for publication Not Applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no conflicts of interest. Funding The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R350), King Saud University, Riyadh, Saudi Arabia Authors' contributions Conceptualization, ; Data curation, ; Formal analys, ; Funding acquisition, ; Investigation, ; Project administration, ; Resources, ; Software, ; Supervision, ; Validation, ; Visualization, ; Writing – original draft, ; Writing – review & editing, References AGARWAL, H., KUMAR, S. V. & RAJESHKUMAR, S. 2017. A review on green synthesis of zinc oxide nanoparticles–An eco-friendly approach. Resource-Efficient Technologies, 3 , 406-413. AGRAWAL, K. V., SHIMIZU, S., DRAHUSHUK, L. W., KILCOYNE, D. & STRANO, M. S. 2017. Observation of extreme phase transition temperatures of water confined inside isolated carbon nanotubes. Nature nanotechnology, 12 , 267-273. AGRAWAL, S. & RATHORE, P. 2014. Nanotechnology pros and cons to agriculture: a review. Int J Curr Microbiol App Sci, 3 , 43-55. AHMAD, N. M., MOHAMED, A. H., ZAINAL-ABIDIN, N., NAWAHWI, M. Z. & AZZEME, A. M. 2024. Effect of optimisation variable and the role of plant extract in the synthesis of nanoparticles using plant-mediated synthesis approaches. Inorganic Chemistry Communications, 161 , 111839. AKBARI, B., TAVANDASHTI, M. P. & ZANDRAHIMI, M. 2011. Particle size characterization of nanoparticles–a practicalapproach. Iranian Journal of Materials Science and Engineering, 8 , 48-56. ALI, S. G., ANSARI, M. A., ALZOHAIRY, M. A., ALOMARY, M. N., JALAL, M., ALYAHYA, S., ASIRI, S. M. M. & KHAN, H. M. 2020. Effect of biosynthesized ZnO nanoparticles on multi-drug resistant Pseudomonas aeruginosa. Antibiotics, 9 , 260. BAER, D. R., GASPAR, D. J., NACHIMUTHU, P., TECHANE, S. D. & CASTNER, D. G. 2010. Application of surface chemical analysis tools for characterization of nanoparticles. Analytical and bioanalytical chemistry, 396 , 983-1002. BORO, B., BORUAH, J. S., DEVI, C., GOGOI, B., BHARALI, P., REDDY, P. V. 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King Saud University, Riyadh, Saudi Arabia, University of Saskatchewan, Saskatoon, SK, Canada, Brunel University London, Uxbridge, United Kingdom, Integral Institute of Medical Sciences and Research, Lucknow, India. KUMAR, A. & DIXIT, C. K. 2017. Methods for characterization of nanoparticles. Advances in nanomedicine for the delivery of therapeutic nucleic acids. Elsevier. KUMARI, S. C., DHAND, V. & PADMA, P. N. 2021. Green synthesis of metallic nanoparticles: a review. Nanomaterials , 259-281. LI, J., SHI, L., ZHANG, X., KANG, X., WEN, Y., QIAN, H., ZHOU, Y., XU, W., ZHANG, Y. & WU, M. 2010. Recombinant adenovirus IL-24-Bax promotes apoptosis of hepatocellular carcinoma cells in vitro and in vivo. Cancer gene therapy, 17 , 771-779. LI, Z., HE, J., LI, B., ZHANG, J., HE, K., DUAN, X., HUANG, R., WU, Z. & XIANG, G. 2020. Titanium dioxide nanoparticles induce endoplasmic reticulum stress-mediated apoptotic cell death in liver cancer cells. 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A Review on Green Synthesis of Nanoparticles and Their Diverse Biomedical and Environmental Applications. Catalysts 2022, 12, 459. s Note: MDPI stays neutral with regard to jurisdictional claims in published …. RAGAVENDRAN, C., KAMARAJ, C., JOTHIMANI, K., PRIYADHARSAN, A., KUMAR, D. A., NATARAJAN, D. & MALAFAIA, G. 2023. Eco-friendly approach for ZnO nanoparticles synthesis and evaluation of its possible antimicrobial, larvicidal and photocatalytic applications. Sustainable Materials and Technologies, 36 , e00597. RAI, R. S., BAJPAI, V., KHAN, M. I., ELBOUGHDIRI, N., SHANABLEH, A. & LUQUE, R. 2023. An eco-friendly approach on green synthesis, bio-engineering applications, and future outlook of ZnO nanomaterial: A critical review. Environmental Research, 221 , 114807. RASTOGI, A., ZIVCAK, M., SYTAR, O., KALAJI, H. M., HE, X., MBARKI, S. & BRESTIC, M. 2017. Impact of metal and metal oxide nanoparticles on plant: a critical review. Frontiers in chemistry, 5 , 78. RATHORE, C., YADAV, V. K., GACEM, A., ABDELRAHIM, S. K., VERMA, R. K., CHUNDAWAT, R. S., GNANAMOORTHY, G., YADAV, K. K., CHOUDHARY, N. & SAHOO, D. K. 2023. Microbial synthesis of titanium dioxide nanoparticles and their importance in wastewater treatment and antimicrobial activities: a review. Frontiers in Microbiology, 14 , 1270245. SHI, H., MAGAYE, R., CASTRANOVA, V. & ZHAO, J. 2013. Titanium dioxide nanoparticles: a review of current toxicological data. Particle and fibre toxicology, 10 , 1-33. SINGH, H., DESIMONE, M. F., PANDYA, S., JASANI, S., GEORGE, N., ADNAN, M., ALDARHAMI, A., BAZAID, A. S. & ALDERHAMI, S. A. 2023. Revisiting the green synthesis of nanoparticles: uncovering influences of plant extracts as reducing agents for enhanced synthesis efficiency and its biomedical applications. International Journal of Nanomedicine , 4727-4750. TIGUNTSEVA, E., CHEBYKIN, A., ISHTEEV, A., HAROLDSON, R., BALACHANDRAN, B., USHAKOVA, E., KOMISSARENKO, F., WANG, H., MILICHKO, V. & TSYPKIN, A. 2017. Resonant silicon nanoparticles for enhancement of light absorption and photoluminescence from hybrid perovskite films and metasurfaces. Nanoscale, 9 , 12486-12493. WANG, F., SJ, P. B. & QIU, W. 2021. Novel oncogenes and tumor suppressor genes in hepatocellular carcinoma. Liver research, 5 , 195-203. XIE, Y., HE, Y., IRWIN, P. L., JIN, T. & SHI, X. 2011. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Applied and environmental microbiology, 77 , 2325-2331. XULU, J. H., NDONGWE, T., EZEALISIJI, K. M., TEMBU, V. J., MNCWANGI, N. P., WITIKA, B. A. & SIWE-NOUNDOU, X. 2022. The use of medicinal plant-derived metallic nanoparticles in theranostics. Pharmaceutics, 14 , 2437. YADAV, R. & AGARWALA, M. 2011. Phytochemical analysis of some medicinal plants. Journal of phytology, 3. ZHANG, X., ZHANG, J., LIU, Y., LI, J., TAN, J. & SONG, Z. 2021. 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Supplementary Files supplementaryfile1.docx Cite Share Download PDF Status: Published Journal Publication published 18 Sep, 2025 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 14 Jul, 2025 Reviews received at journal 06 Jul, 2025 Reviewers agreed at journal 02 Jul, 2025 Reviews received at journal 28 Jun, 2025 Reviewers agreed at journal 28 Jun, 2025 Reviewers agreed at journal 28 Jun, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 27 Jun, 2025 Reviewers invited by journal 26 Jun, 2025 Editor assigned by journal 26 Jun, 2025 Submission checks completed at journal 26 Jun, 2025 First submitted to journal 24 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6961371","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":477811524,"identity":"a0da2cdf-7e2d-43af-b1c1-629c156c4ac5","order_by":0,"name":"Tayyab Shafiq","email":"","orcid":"","institution":"the First Affiliated Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Tayyab","middleName":"","lastName":"Shafiq","suffix":""},{"id":477811525,"identity":"ea5fb88a-f801-49f4-833d-96a97009437e","order_by":1,"name":"Humaira Yasmin","email":"","orcid":"","institution":"Comsats University","correspondingAuthor":false,"prefix":"","firstName":"Humaira","middleName":"","lastName":"Yasmin","suffix":""},{"id":477811526,"identity":"6e1d5cbe-a4a9-45fc-8ad1-05dcabd2870f","order_by":2,"name":"Li Duan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYDCCA0D8oALCliBeS8IZkrUktpGihe9G8rMHifPq7A0OMB+8zcNgl0dQi+SNNHODxG2HmQ0OsCVb8zAkFxPUYnAjwUwicdsBNoMDPGbSPAwHEhsIa0n/JpE4p47H4AD/N2K15ABtaWCWANrCRpwWyTNvyiQSjh02kDzMZmw5xyCZsBa+4+nbJD7U1NnzHW9+eONNhR1hLQjADHYn8epHwSgYBaNgFOABAMNROb12dBdiAAAAAElFTkSuQmCC","orcid":"","institution":"the First Affiliated Hospital of Shenzhen University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Duan","suffix":""},{"id":477811527,"identity":"b08e628c-9a7e-41b9-bb94-9e5d7b94ca4c","order_by":3,"name":"Nukhbat Ullah","email":"","orcid":"","institution":"King Edward Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nukhbat","middleName":"","lastName":"Ullah","suffix":""},{"id":477811528,"identity":"b50dddee-777f-4db5-8fa6-1b5872e4a881","order_by":4,"name":"Tariq Nadeem","email":"","orcid":"","institution":"University of The Punjab","correspondingAuthor":false,"prefix":"","firstName":"Tariq","middleName":"","lastName":"Nadeem","suffix":""},{"id":477811529,"identity":"4f5b26ee-9b2a-4767-96cd-a71f57f1ec89","order_by":5,"name":"Mohsin Zafar","email":"","orcid":"","institution":"Comsats University","correspondingAuthor":false,"prefix":"","firstName":"Mohsin","middleName":"","lastName":"Zafar","suffix":""},{"id":477811530,"identity":"066e4baf-8e7f-4fc1-99ed-2cbb655a1d8d","order_by":6,"name":"Nadeem Hassan","email":"","orcid":"","institution":"Comsats University","correspondingAuthor":false,"prefix":"","firstName":"Nadeem","middleName":"","lastName":"Hassan","suffix":""},{"id":477811531,"identity":"5683b97b-3895-4b3f-b65d-71b380165ecf","order_by":7,"name":"Ajaz Ahmad","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Ajaz","middleName":"","lastName":"Ahmad","suffix":""},{"id":477811532,"identity":"a91291d7-c322-4e58-95a0-1ccbbfc96fc0","order_by":8,"name":"Usama Ahmed","email":"","orcid":"","institution":"Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Usama","middleName":"","lastName":"Ahmed","suffix":""}],"badges":[],"createdAt":"2025-06-24 04:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6961371/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6961371/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02154-4","type":"published","date":"2025-09-18T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85687755,"identity":"7fd3cc96-c5cd-4888-810a-bfd6e0343dbe","added_by":"auto","created_at":"2025-06-30 16:19:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":417884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;nanoparticles (NPs) synthesized using \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCinnamomum verum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e bark extract.\u003c/strong\u003e (a) Optimization of plant extract quantity shows the best results with 20 ml of extract for both CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;NPs. (b) Optimization of stirring time indicates that 120 minutes is optimal for nanoparticle synthesis. (c) Temperature optimization reveals that CV-ZnO \u0026nbsp;NPs are optimally synthesized at 70°C, while CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;NPs at 40°C. (d) pH optimization shows that a pH of 7 is optimal for the synthesis of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;NPs. (e) Salt stability test indicates that CV-TiO\u003csub\u003e2 \u0026nbsp;\u003c/sub\u003e(blue) and CV-ZnO \u0026nbsp;(orange) NPs remain stable at 1, 2, and 3 M concentrations of sodium chloride.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/ca64aa62244dc29538def863.jpeg"},{"id":85689050,"identity":"2c3cb67c-9eec-4c3d-8ff9-960077ee81bc","added_by":"auto","created_at":"2025-06-30 16:35:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":304084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;NPs synthesized using \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCinnamomum verum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e bark extract.\u003c/strong\u003e (a) UV-Visible spectrophotometry analysis showing the UV-Vis spectrum of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;NPs with peak 300-400 nm and CV-ZnO \u0026nbsp;NPs with a peak around 300-320 nm, (b) FTIR spectrum of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles showing characteristic peaks for O-H, C-H stretching and C-H bending, (c) FTIR spectrum of CV-ZnO \u0026nbsp;nanoparticles displaying peaks for medium O-H stretching, O=C=O stretching, and C-H bending, (d) XRD pattern of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles showing distinct peaks at 25.3°, 37.8°, 48.0°, 54.1°, and 62.7°, (e) XRD pattern of CV-ZnO \u0026nbsp;nanoparticles displaying peaks at 31.7°, 34.4°, 36.3°, 47.5°, and 56.6°.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/9c467fbabe2d4203afa46eec.jpeg"},{"id":85687757,"identity":"c60b6f16-090b-4967-b8fe-c1c795b8a9a6","added_by":"auto","created_at":"2025-06-30 16:19:32","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":316232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning Electron Microscopy (SEM) analysis of CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;NPs at 10 µm resolution.\u003c/strong\u003e (a) SEM image of CV-TiO2 \u0026nbsp;NPs showing spherical shapes and fused agglomerates, (b) SEM image of CV-ZnO \u0026nbsp;NPs displaying spherical shapes and fused agglomerates.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/61929e0d790f80ae82b4413c.jpeg"},{"id":85687756,"identity":"99095de4-e661-428a-918e-2020e4b798d1","added_by":"auto","created_at":"2025-06-30 16:19:32","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPPH radical scavenging assay to evaluate the antioxidant activity of CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;nanoparticles.\u003c/strong\u003e The assay was performed at various concentrations (5, 10, 25, 50, 100 µg/ml). (a) Antioxidant activity of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles, (b) Antioxidant activity of CV-ZnO \u0026nbsp;nanoparticles, (C) Comparative antioxidant activities of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eand CV-ZnO \u0026nbsp;at same concentrations.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/8fd9efd8c5fc13ef48a47fa6.jpeg"},{"id":85688709,"identity":"ff96e9d9-4c41-47ae-8fc1-b0370889a9ee","added_by":"auto","created_at":"2025-06-30 16:27:32","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePercent SOD activity of CV-ZnO \u0026nbsp;and CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;nanoparticles treated Huh-7 cells. \u003c/strong\u003e(a) SOD activity of Huh-7 cells treated with CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;NPs, (b) SOD activity of Huh-7 cells treated with CV-ZnO \u0026nbsp;NPs.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/f68f13a516848f0fc1cbc821.jpeg"},{"id":85688712,"identity":"d8f02939-0320-4bc0-856f-4f89966a673e","added_by":"auto","created_at":"2025-06-30 16:27:32","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":114300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat maps showing the antibacterial activity of CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;nanoparticles against different bacterial strains (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cereus, S. aureus, E. coli, E. aerogenes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e). \u003c/strong\u003eThe antibacterial activity was evaluated using the disc diffusion method at various concentrations (5, 10, 15, 20, 30, 50, 100, and 150 ppm). (a) Heat map for CV-TiO2 \u0026nbsp;nanoparticles displaying zones of inhibition (b) Heat map for CV-ZnO \u0026nbsp;nanoparticles showing zones of inhibition for the same bacterial strains. The color intensity indicates the level of antibacterial activity, with darker red colors representing larger zones of inhibition.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/51c60921938d1ec3de0222d4.jpeg"},{"id":85688727,"identity":"58189aaa-ba9f-479e-8a9f-8a9476ab9a49","added_by":"auto","created_at":"2025-06-30 16:27:33","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":92254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat maps illustrating the antifungal activity of CV-TiO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u0026nbsp;and CV-ZnO \u0026nbsp;nanoparticles against various fungal strains \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e(Alternaria solan, Microphobia phacelia, Aspergillus Niger, Candida albicans)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e The antifungal activity was assessed using the disc diffusion method at different nanoparticle concentrations (50, 100, 150, and 200 ppm). (a) Heat map for CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles showing zones of inhibition against fungal strains (b) Heat map for CV-ZnO \u0026nbsp;nanoparticles displaying zones of inhibition for the same fungal strains. The color intensity represents the level of antifungal activity, with red darker colors indicating larger zones of inhibition.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/a764be9296bdc7bce1a6bcf3.jpeg"},{"id":85687765,"identity":"ccf2490a-3128-49bd-8b44-91a1d4587011","added_by":"auto","created_at":"2025-06-30 16:19:32","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":87579,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity assessment of CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles at incremental doses against Huh-7 liver cancer cells incubated for 48 hours. The experiment was performed in triplicates. (a) Cytotoxicity profile of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles (b) Cytotoxicity profile of CV-ZnO \u0026nbsp;nanoparticles.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/73897d964267114c66f661fb.jpeg"},{"id":85687767,"identity":"d9623923-ced3-499b-ba34-7e0186afd7b5","added_by":"auto","created_at":"2025-06-30 16:19:32","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":84269,"visible":true,"origin":"","legend":"\u003cp\u003eThe RT-PCR based mRNA expression studies of inflammatory and apoptotic marker genes in Huh-7 liver cancer cells treated with CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003enanoparticles. GAPDH was used as an internal control. a) highlights mRNA expression of AFP b) indicates mRNA levels of Bax c) shows fold change of Bcl-2 d) reflects PTEN expression in fold change in comparison with the control.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/b83fa54953cbf9d1580fa13c.jpeg"},{"id":91889868,"identity":"a14f644d-4599-4035-b90e-87e6624be8cf","added_by":"auto","created_at":"2025-09-22 16:02:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3024153,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/cf46d088-e1e7-4016-ba2b-5726a77389de.pdf"},{"id":85690104,"identity":"b2fe30e9-333b-4204-bb42-3e85bbbda8ba","added_by":"auto","created_at":"2025-06-30 16:43:32","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1679335,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6961371/v1/7f3c2e68c62ec4901b66e40c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zinc Oxide and Titanium Dioxide Nanoparticles Bio fabricated for Enhanced Antimicrobic, Antioxidant, and Antitumor Performance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanotechnology has embarked multidisciplinary applications in the field of research with profound developments. The synthesis of metal and metal oxide nanoparticles (NPs) involves the use of depleting and balancing agents to produce materials with distinct characteristics (Rastogi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (Agarwal et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Plants are reported to be capable of reducing metal ions. Many factors influence the synthesis of nanoparticles, including pH, temperature, and reaction time (Tiguntseva et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (Agrawal and Rathore, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCV-ZnO has been categorized as a safe metal oxide by the FDA (Agrawal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is used in a variety of consumer items, including lotions, ceramics, wastewater treatment, and rubber processing. CV-ZnO absorbs UV radiation and is consequently used in cosmetics. The antibacterial properties make it odour-resistant, and its anticancer properties render it a promising tumour therapy option (Agrawal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Agarwal et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTitanium dioxide (CV-TiO\u003csub\u003e2\u003c/sub\u003e ) nanoparticles have long been regarded as low-toxicity, weakly soluble particles. They are used as a negative control in particle toxicology research, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. In recent years, CV-TiO\u003csub\u003e2\u003c/sub\u003e NPs have become increasingly popular in industrial and consumer applications due to their larger surface area per unit mass and catalytic activity. Recent studies suggest that CV-TiO\u003csub\u003e2\u003c/sub\u003e NPs exhibit different bioactivities (Shi et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A variety of plants have been reported to benefit from titanium dioxide nanoparticles (CV-TiO\u003csub\u003e2\u003c/sub\u003e ), benefiting sustainable agriculture by reducing soil salinity. They are used as a foliar spray to boost plant growth enzyme activity, chlorophyll content photosynthesis, nutrient absorption, stress tolerance, yield, and crop quality. They poesses small size, easy handling, long-term storage, high efficacy, and nontoxicity (El-Said et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCinnamon is a potent spice that has been used medicinally for thousands of years. Cinnamon ranks first among twenty-six of the world's most popular herbs and medicinal spices due to its beneficial antioxidant levels. Cinnamon bark extract is a key bioactive component with a variety of biological activities, including antibacterial, antibiofilm, anthelmintic, anticancer, and antifungal properties (Ali et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eC. verum\u003c/em\u003e bark extract can be used to synthesize CV-ZnO and CV-TiO\u003csub\u003e2\u003c/sub\u003e NPs. In the current investigation, the biogenic one-step synthesis and capping of Zinc and titanium nanoparticles using cinnamon bark extract was carried out. Green synthesis of metallic nanoparticles has several advantages over chemical and physical production, including decreased toxicity, environmental friendliness, low energy consumption, and cost-effectiveness (Ali et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntimicrobial resistance is a growing global concern. Current medicinal practices and the widespread use of broad-spectrum antibiotics have exacerbated the raised concern. Similarly, antifungal resistance poses significant challenges, particularly for patients with invasive fungal infections affecting critical organs. Addressing these issues necessitates innovative approaches. Furthermore, the cytotoxic effects of metallic nanoparticles against cancer cell lines highlight their potential in cancer treatment. Due to their distinct physicochemical characteristics and versatile applications, nanoparticles are potential drug-delivery vehicles to treat cancer. (Lee et al., 2006).\u003c/p\u003e \u003cp\u003eIn the current investigation, we explored the promising potential of green synthesized bio-fabricated zinc oxide (CV-ZnO ) and titanium oxide (CV-TiO\u003csub\u003e2\u003c/sub\u003e ) nanoparticles as multifunctional materials with significant implications in combating human pathogens and exhibiting anti-cancerous activities. The synthesized nano biocomposites were characterized using various techniques, including XRD, FTIR, SEM and UV spectroscopy. Their antibacterial potential was assessed against four pathogenic bacterial strains, including \u003cem\u003eBacillus cereus, Staphylococcus aureus Enterobacter aerogenes\u003c/em\u003e and \u003cem\u003eEscherichia coli.\u003c/em\u003e The antifungal potnential was evaluated against \u003cem\u003eAlternaria solani, Macrophomina phaseolina, Aspergillus niger, Candida albicans\u003c/em\u003e. \u003cem\u003eIn vitro\u003c/em\u003e anticancer activity of these nano biocomposites was examined against the Huh-7 liver cancer cell line.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eSynthesis of Nanoparticles and Protocol Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBark samples of Cinnamomum verum were initially rinsed with distilled water thrice and allowed to dry overnight. Following this, 30 g of bark was ground into a fine powder, and 20 g of the powder was added to 100 ml of water and heated on a hot plate for approximately 30 minutes at 50-60\u0026deg;C until the solution turned dark brown. The extract was then filtered using Whatman no. 1 filter paper to remove impurities, frozen, and stored at 5\u0026deg;C for future use. For the synthesis of CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles, two salt solutions were prepared at different concentrations. A 0.1 M solution of zinc nitrate (Sigma Aldrich, Zn(NO3)2\u0026middot;6H2O, CAS Number 10196-18-6) was prepared by dissolving 14.873 g of zinc nitrate in 500 ml of distilled water. Similarly, a 1 M solution of titanium chloride (Sigma Aldrich, TiCl4, CAS No: 7550-45-0) was prepared by dissolving 18.9 ml of titanium chloride in 100 ml of distilled water. Both solutions were stirred for two minutes using a magnetic stirrer to ensure complete dissolution and stored in 500 ml beakers. To achieve optimal synthesis conditions for CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs, various parameters were optimized, including the quantity of plant extract, stirring time, reaction temperature, pH of the solution, and salt stability. The reaction mixture was then centrifuged at 5000 rpm for 5 minutes to obtain the nanoparticles. The supernatant was discarded, and the pellet was retained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of CV-TiO2 \u0026nbsp;and CV-ZnO \u0026nbsp;NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of the synthesized CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;nanoparticles was performed using various techniques to determine their structural, optical, and morphological properties.\u003c/p\u003e\n\u003cp\u003eUV-visible spectrophotometry: This technique was used to investigate the optical properties of the synthesized nanoparticles. The absorption spectra of the CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs were recorded over a wavelength range of 200-1000 nm using a UV-Vis spectrophotometer. The samples were prepared by dispersing the nanoparticles in distilled water and sonicating them for 30 minutes to ensure a uniform suspension. The absorbance was measured.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Fourier Transform Infrared Spectroscopy: FTIR spectroscopy was used to identify the functional groups and capping agents present on the surface of the nanoparticles. The FTIR spectra of the CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs were recorded. The samples were prepared by mixing the nanoparticles with potassium bromide (KBr) to form pellets. The spectra were obtained in the range of 4000-400 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eX-Ray Diffraction (XRD): This method was conducted to determine the crystalline structure, phase purity, and average crystalline size of the synthesized nanoparticles. The XRD patterns of the CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs were obtained using an X-ray diffractometer equipped with Cu K\u0026alpha; radiation (\u0026lambda; = 1.5406 \u0026Aring;). The samples were prepared by placing the nanoparticles on a glass slide. The data were collected over a 2\u0026theta; range of 10\u0026deg; to 80\u0026deg; with a step size of 0.02\u0026deg;.\u003c/p\u003e\n\u003cp\u003eScanning Electron Microscopy: This technique was used to examine the surface morphology, size distribution, and overall shape of the nanoparticles. The SEM images of the CV-TiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e and CV-ZnO \u0026nbsp;NPs were captured using a scanning electron microscope. The samples were prepared by placing a drop of nanoparticle suspension onto a silicon wafer, followed by drying at room temperature. The dried samples were coated with a thin layer of gold to enhance conductivity before imaging.\u003c/p\u003e\n\u003cp id=\"_Toc91451894\"\u003eBiomedical Applications of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe synthesized CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs were evaluated for their antimicrobial, antifungal, and antioxidant activities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial Activity:\u003c/strong\u003e The antimicrobial ability of the NPs was determined against four pathogenic bacterial strains, including \u003cem\u003eBacillus cereus,\u003c/em\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e \u003cem\u003eEnterobacter aerogenes\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Escherichia coli,\u003c/em\u003e using the disc diffusion method. To assess the antibacterial activity of CV-TiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e and CV-ZnO \u0026nbsp;nanoparticles (NPs), seven different concentrations were prepared (5, 10, 15, 20, 30, 50, 100, and 150 ppm). The suspensions of CV-ZnO \u0026nbsp;NPs in water were sonicated for 20-30 minutes at 60\u0026deg;C to ensure complete solubilization of the NPs. The same procedure was for CV-TiO\u003csub\u003e2\u003c/sub\u003e NPs but with DMSO. The discs were soaked for approximately 20 minutes with varying concentrations of NPs and dried. Autoclaved media was poured into Petri plates and allowed to solidify for about an hour. A 10 \u0026micro;L concentration of bacterial strains from LB media was added to each Petri plate. The culture was evenly spread across the plate using a spreader. This procedure was performed for each bacterial strain, with three replicates for each strain. The sealed plates were placed in an incubator at room temperature for 24-48 hours. The plates were examined for the bactericidal activity of the NPs. The zones of inhibition around the discs were measured to determine the antibacterial efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntifungal Activity:\u003c/strong\u003e The antifungal activity of the NPs was tested against \u003cem\u003eAlternaria solani\u003c/em\u003e, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e, \u003cem\u003eAspergillus niger\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e using the disc diffusion method. The procedure was similar to the antibacterial activity assay, with different nanoparticle concentrations tested for antifungal efficacy (30, 50, 100 and 150 ppm). Water was used as a control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDPPH Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant activity was assessed using the DPPH radical scavenging assay. Various concentrations of the nanoparticle samples were mixed with 0.4 mM DPPH in ethanol, and the reaction mixtures were incubated for 30 minutes at room temperature. Absorbance was measured at 517 nm, and the percentage of radical scavenging activity was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuperoxide dismutase (SOD) Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the manufacturer\u0026apos;s instructions, the SOD assay kit was used to assess the SOD activity in Huh-7 cells treated with CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles. For each well, 20 \u0026mu;L of the supernatant from different experimental groups was added, followed by the addition of 200 \u0026mu;L of WST solution. This included blanks 1, 2, and 3 as reference points. Subsequently, 20 \u0026mu;L of the enzyme working solution was added to all wells containing the samples and blank 1. The plate was gently mixed and incubated at 37 \u0026deg;C for 20 minutes. After incubation, the absorbance was measured at 450 nm using a microtiter plate reader. To ensure reproducibility, all samples were analyzed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity Assessment Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the cytotoxic potential of CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles synthesized in the current investigation against Huh-7 liver cancer cells. To assess the cytotoxicity, a total of 5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e cells were seeded in 96-well flat-bottom plates containing DMEM supplemented with 5% FBS. Subsequently, various concentrations of CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles (5, 10, 25, 50, 100 \u0026mu;g/mL) were added to the wells. The cells were incubated for 48 hours at 37 \u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. After that, the cell monolayers were rinsed with serum-free media, and 100 \u0026mu;L of a 5 mg/mL MTT solution was added to each well. The plates were incubated for 4\u0026ndash;5 hours to allow the MTT into formazan crystals by viable cells. Following this, the media was aspirated, and the formazan crystals were dissolved in 100 \u0026mu;L of 0.1% DMSO. The absorbance of the resulting solution was measured at 550 nm using a microplate reader (Multiskan GO, Thermo Scientific, USA). In parallel, control cells treated with PBS buffer instead of green synthesized nanoparticles were taken as a negative control. The experiment was performed in triplicate. The percentage of cell inhibition was calculated using the formula [(Ac\u0026ndash;As)/(Ac)] *100, where Ac represents the absorbance of the control wells, and As represents the absorbance of the sample wells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR-based expression profiling of proinflammatory and apoptotic genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuh-7 cells were incubated in six-well plates for a duration of 24 hours, followed by treatment with 50 and 100 \u0026micro;g/ml of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles. The purpose of this treatment was to isolate total RNA from the cells. To extract the RNA, Trizol was utilized in accordance with the manufacturer\u0026apos;s instructions. The samples were collected from both control cells and those treated with stress and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles. The concentration of the extracted RNA was determined by measuring the absorbance at 260 nm using a Nanodrop spectrophotometer. After ethanol washing (1 mL), the RNA was dissolved in 50 \u0026micro;L of 0.1% Diethyl pyrocarbonate (DEPC) treated water and stored at \u0026minus;80 \u0026deg;C until further use. For reverse transcription, the RevertAidTM first-strand synthesis kit (Thermo Scientific, Cat No: K1622) was used. Following the manufacturer\u0026apos;s instructions, 1 \u0026micro;g of RNA was reverse transcribed into cDNA. Forward and reverse primers (0.5 \u0026micro;M each) specific to the genes of interest (AFP, Bax, Bcl-2, and PTEN) (Wang et al., 2021) were used in the study. Template cDNA (2 \u0026mu;L) was added to a final reaction volume of 20 \u0026micro;L. Real-time PCR was performed using a StepOne Plus thermocycler (Applied Biosystems) and SYBR Green PCR Master Mix (Thermofisher, catalogue number K0221). All real-time PCR assays were run in triplicate. The data were reported as the mean of three independent experiments. The transcriptomic expression of the selected genes was normalized with the GAPDH gene used as an internal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach experimental assay was performed in triplicate. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc, La Jolla, CA). The significance level for determining differences between mean values was set at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eOptimization of Green Synthesis of CV-TiO\u003csub\u003e2\u003c/sub\u003e and CV-ZnO \u0026nbsp;NPs\u003c/h2\u003e\n\u003cp\u003eFigure 1a shows the effect of varying the quantity of plant extract on the synthesis of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs. \u0026nbsp;Optimal synthesis was achieved using 20 ml of plant extract for both CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs with optimal size and stability. Figure 1b presents the effect of different stirring times on nanoparticle synthesis. The optimal stirring time for CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs was found to be 120 minutes. This duration allowed for adequate interaction between the extract and the metal salts. CV-TiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e NPs showed optimal synthesis at 40\u0026deg;C, while CV-ZnO \u0026nbsp;NPs showed the best results at 70\u0026deg;C (Figure 1c). These temperatures facilitated the efficient conversion of metal salts to stable and uniformly sized NPs. CV-TiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e and CV-ZnO \u0026nbsp;NPs showed optimal synthesis at a pH of approximately 7 (Figure 1d). The optimal pH level ensured the proper ionization of the bioactive compounds in the extract, enhancing their reducing capability. Both nanoparticles remained stable under all tested salt concentrations (Figure 1e).\u003c/p\u003e\n\u003ch2\u003eCharacterization of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs\u003c/h2\u003e\n\u003ch3 id=\"_Toc91451925\"\u003eUV-Vis Spectrophotometry\u003c/h3\u003e\n\u003cp\u003eMaximum absorbance peak was observed between 300-400 nm during the synthesis of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs and around 300-320 nm during the synthesis of CV-ZnO \u0026nbsp;NPs (Figure 2a). These peaks reflect their respective characteristic band gap absorption of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs.\u003c/p\u003e\n\u003ch3 id=\"_Toc91451926\"\u003eFourier Transforms Infra-Red Spectroscopy (FTIR):\u003c/h3\u003e\n\u003cp\u003eThe FTIR spectrum of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eNPs (Figure 2b) indicates that there are various stretches at different levels giving different peaks (3550-3200 cm\u003csup\u003e-1\u003c/sup\u003e strong O-H stretching, 3000-2840 cm\u003csup\u003e-1\u003c/sup\u003e medium C-H stretching, 2000-1650 cm\u003csup\u003e-1\u003c/sup\u003e weak C-H bending, 1000-650 cm\u003csup\u003e-1\u003c/sup\u003e strong C-C bending, and 900-700 strong C-H bending). In the case of CV-ZnO \u0026nbsp;NPs, slightly different vibration peaks were observed, including 3700-3584 cm\u003csup\u003e-1\u003c/sup\u003e medium O-H stretching, 2400-2000 cm\u003csup\u003e-1\u003c/sup\u003e strong O=C=O stretching, and 2000-1650 cm\u003csup\u003e-1\u003c/sup\u003e weak C-H bending (Figure 2c). These peaks confirm the presence of functional groups involved in the stabilization and capping of both NPs.\u003c/p\u003e\n\u003ch3\u003eX-Ray Diffraction\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe XRD pattern of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs reveals the distinct peaks at 25.3\u0026deg;, 37.8\u0026deg;, 48.0\u0026deg;, 54.1\u0026deg;, and 62.7\u0026deg;, corresponding to the anatase phase of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003e(Figure 2d). These peaks confirm the crystalline nature and phase purity of the synthesized CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles. The CV-ZnO \u0026nbsp;nanoparticles exhibited peaks at 31.7\u0026deg;, 34.4\u0026deg;, 36.3\u0026deg;, 47.5\u0026deg;, and 56.6\u0026deg;, characteristic of the wurtzite hexagonal phase of CV-ZnO \u0026nbsp;(Figure 2e). These peaks indicate the crystallinity and phase purity of the CV-ZnO \u0026nbsp;nanoparticles.\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM)\u003c/h3\u003e\n\u003cp\u003eThe SEM images were obtained at 10 \u0026micro;m resolution. The synthesized NPs possessed uniformly dispersed spherical shapes and fused agglomerates. Their uniform dispersion and spherical morphology indicate a narrow size distribution. CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs were seen uniformly dispersed with a size ranging from 40 nm to 80 nm (Figure 3a). The SEM image of CV-ZnO \u0026nbsp;NPs sizes also ranged from 40 nm to 80 nm with similar uniform dispersion (Figure 3b).\u003c/p\u003e\n\u003ch3\u003eAntioxidant activity of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eDPPH Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eand CV-ZnO \u0026nbsp;NPs to scavenge free radicals was evaluated by using a DPPH assay. The results show a concentration-dependent increase in antioxidant activity of both nanoparticles (Figure 4). The scavenging activity of CV-ZnO \u0026nbsp;NPs was slightly lower than that of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs at higher concentrations (Figure 4c). At 50 \u0026micro;g/ml, the CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eNPs showed a maximum activity of 90% (Figure 4a). In the case of CV-ZnO \u0026nbsp;NPs, it was 50% activity at the same concentration (Figure 4b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOD Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide Dismutase (SOD) is responsible for the conversion of superoxide ions into less harmful byproducts. The activity of this enzyme was examined in Huh-7 cells treated with CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles. The SOD activity was found to be higher in the CV-TiO\u003csub\u003e2\u003c/sub\u003e -treated cells compared to the CV-ZnO -treated cells at 25, 50, and 100 \u0026mu;g/ml concentrations (Figure 5). The percent SOD activity in Huh-7 cells treated with CV-ZnO \u0026nbsp;nanoparticles at these concentrations was 34.94%, 46.16%, and 49.02%, respectively. In contrast, the percent SOD activity in Huh-7 cells treated with CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles was 42.76%, 53.34%, and 59.73%, respectively.\u003c/p\u003e\n\u003ch3\u003eAntibacterial Activity of CV-TiO\u003csub\u003e2\u003c/sub\u003e , CV-ZnO \u0026nbsp;NPs\u003c/h3\u003e\n\u003cp\u003eAntibacterial activity of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs was observed against pathogenic bacterial strains (\u003cem\u003eB. cereus, S. aureus, E. coli, E. aerogenes\u003c/em\u003e) as shown in Figure 6. CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eNPs (200 ppm) showed 86% inhibitions against \u003cem\u003eB. cereus,\u0026nbsp;\u003c/em\u003e80% inhibition against\u003cem\u003e\u0026nbsp;S. aureus,\u0026nbsp;\u003c/em\u003e74% against \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand 71% against \u003cem\u003eE. aerogenes\u0026nbsp;\u003c/em\u003e(Figure 6a)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eNon-significant percent inhibition was observed at 5 ppm against these bacterial strains. Similarly, CV-ZnO \u0026nbsp;NPs at 200 ppm showed 76% inhibition against \u003cem\u003eB. cereus,\u0026nbsp;\u003c/em\u003e75% against\u003cem\u003e\u0026nbsp;S. aureus,\u0026nbsp;\u003c/em\u003e74% against \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand 71% against \u003cem\u003eE. aerogenes\u0026nbsp;\u003c/em\u003e(Figure 6b)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eCV-ZnO \u0026nbsp;NPs exhibited non-significant percent inhibition at 5 ppm concentration against these bacterial strains. Antibacterial activity of plant extract of \u003cem\u003ecinnamon verum\u0026nbsp;\u003c/em\u003eshowed 36% inhibition against \u003cem\u003eB. cereus,\u0026nbsp;\u003c/em\u003e34% inhibition against\u003cem\u003e\u0026nbsp;S. aureus,\u0026nbsp;\u003c/em\u003e24% against \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand 22% against \u003cem\u003eE. aerogenes.\u003c/em\u003e The antibiotic (ciprofloxacin) showed 56% inhibition against \u003cem\u003eB. cereus,\u0026nbsp;\u003c/em\u003e51% inhibition against\u003cem\u003e\u0026nbsp;S. aureus,\u0026nbsp;\u003c/em\u003e61% against E\u003cem\u003e. coli\u0026nbsp;\u003c/em\u003eand 61% against \u003cem\u003eE. aerogenes\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eAntifungal Activity of CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; and CV-ZnO \u0026nbsp;NPs\u003c/h3\u003e\n\u003cp\u003eCV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs at 200 ppm showed 28% inhibition against \u003cem\u003eAlternaria solani\u003c/em\u003e, 26% inhibition against \u003cem\u003eAspergillus niger,\u003c/em\u003e 22% against \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e and 21% against \u003cem\u003eCandida albicans\u003c/em\u003e. At 50 ppm concentration, CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; NPs showed no percent inhibition against any fungal strains (Figure 7a). CV-ZnO \u0026nbsp;NPs at 200ppm showed 23% inhibition against \u003cem\u003eAlternaria solani\u003c/em\u003e, 21% inhibition against \u003cem\u003eAspergillus niger,\u003c/em\u003e 20% against \u003cem\u003eMacrophomina phacelia\u003c/em\u003e and 19% against \u003cem\u003eCandida albicans\u003c/em\u003e. At 50 ppm concentration, they showed no percentage inhibition against any fungal strain (Figure 7b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity Assessment of CV-TiO\u003csub\u003e2\u003c/sub\u003e \u003csub\u003e\u0026nbsp;\u003c/sub\u003eand CV-ZnO \u0026nbsp;NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 8 depicts the impact of different concentrations of CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles on the proliferation of Huh-7 cancer cells. Notably, increasing the concentration of nanoparticles led to a decrease in cancer cell viability. CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles displayed a higher cytotoxic response compared to CV-ZnO \u0026nbsp;nanoparticles across all concentrations. At concentrations of 50 \u0026mu;g/ml and 100 \u0026mu;g/ml of CV-ZnO \u0026nbsp;nanoparticles, the cell viability decreased to 55.3% and 43.68%, respectively. Conversely, Huh-7 cells treated with CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles exhibited a cell viability of 44.17% and 32.329% at the respective concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative RT-PCR Analysis For Apoptotic And Antiapoptotic Markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles exhibited better radical scavenging activity, SOD activity and cytotoxicity than CV-ZnO . As a result, the quantitative RT-PCR analysis was performed for Huh-7 cells treated with CV-TiO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e nanoparticles to assess the mRNA expression levels of inflammation and apoptosis-associated genes (Figure 9). Proinflammatory cytokines (AFP, Bcl-2, and PTEN) were significantly elevated in the stress groups after stimulation with cobalt chloride. Green synthesized CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; nanoparticles significantly lowered the levels of these gene expressions in a dose-dependent manner compared to the stress group (Figures 9a, 9c, and 9d). The mRNA levels of the apoptotic gene (Bax) in Huh-7 cells treated with \u0026nbsp;CV-TiO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; resulted in its upregulation (Figure 9b).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMetallic nanoparticles exhibit potent antimicrobial, antifungal and anticancer potential (Zhu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Zhu et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Kumari et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Baer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, we synthesized CV-TiO2 and CV-ZnO NPs (Chung et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) with the bark extract of \u003cem\u003eCinnamon Verum\u003c/em\u003e. Plant phytochemicals, including alkaloids and flavonoids, play a significant role in reducing the metallic salts and the nanoparticles are precipitated at the end of the reaction as byproducts (Yadav and Agarwala, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the above data, it can be stated that the optimal volume of Cinnamomum verum bark extract to be used is 20 ml, while lesser volumes of the extract led to poor formation of nanoparticles. This is quite in tandem with (Nazir et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) that stated that 15-25ml of plant extract was required in order to get the right bioactive content that will help in reduction and stabilisation of CV-ZnO nanoparticles Likewise, (Ahmad et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (Ragavendran et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) established that adequate stirring was attained in 30 minutes to enhance the interfacial area for the synthesis of CV-ZnO nanoparticles Stated (Rathore et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed that 45 minutes of stirring was appropriate to ensure the proper distribution and stability of the CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles. Secondly, the current investigation specified the reaction temperature of form CV-ZnO nanoparticles to be 70\u0026deg;C and that of CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles to be 40\u0026deg;C. This supports (Rai et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) who postulated that the synthesis of CV-ZnO nanoparticles was most favorable at around 60- 75oC, and (Singh et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) who noted that synthesis of CV-TiO2 nanoparticles preferred at 40\u0026ndash;50oC. Pretreatment of the plant extract was carried out at a neutral pH because it was important to achieve the optimal ionization of some of the compounds in the plant extracts to improve their ability to reduce and stabilize the metal ions loading into the reaction; this is in agreement with (Boro et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) who postulated that neutral pH should be used in the synthesis process of CV-ZnO and CV-TiO\u003csub\u003e2\u003c/sub\u003e Finally, our work pointed out that nanoparticle stability in saline conditions could be crucial if the nanoparticles are to be used in a biomediocre setting. Following the same line, (Patel et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) concluded that CV-ZnO and CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles produced from plant extracts had great stability in saline conditions for physiological uses. The amount of plant extract used in the synthesis process is critical to achieve nanoparticles with desirable characteristics. Our study tested various quantities of \u003cem\u003eC. verum\u003c/em\u003e bark extract. The optimal quantity (20 ml) provided sufficient bioactive compounds necessary for the reduction and stabilization of the metal ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Insufficient amounts of extract resulted in suboptimal nanoparticle formation. The stirring time during the synthesis process significantly affects the interaction between the metal ions and the bioactive compounds in the plant extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The reaction temperature is another crucial factor that influences the rate of reaction and the quality of the synthesized nanoparticles. The optimal temperature for ZnO NPs synthesis was found to be 70\u0026deg;C, while for TiO2 NPs, it was 40\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Maintaining a neutral pH ensured the proper ionization of the plant extract compounds, enhancing their reducing and stabilizing capabilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The stability of nanoparticles in saline environments is essential for their potential biomedical applications. This resilience makes them suitable for applications in physiological environments where salt concentrations vary (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eNanomaterials are characterized using a variety of techniques (Akbari et al., 2011) (Baer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) (Kumar and Dixit, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For CV-TiO\u003csub\u003e2\u003c/sub\u003e NPs, a characteristic absorbance peak shows the anatase phase of CV-TiO2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This peak is indicative of the band gap absorption. The sharp and distinct peaks in the UV-Vis spectra affirm the successful synthesis of both NPs. The FTIR spectrum of CV-TiO\u003csub\u003e2\u003c/sub\u003e and CV-ZnO NPs displayed characteristic peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This highlights the effective capping and stabilization of the nanoparticles by the bioactive compounds in the \u003cem\u003eC. verum\u003c/em\u003e bark extract containing hydroxyl and organic groups (Xulu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The XRD pattern of CV-TiO\u003csub\u003e2\u003c/sub\u003e and CV-ZnO NPs revealed distinct peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). These sharp peaks confirm the high crystallinity and phase purity of the CV-TiO\u003csub\u003e2\u003c/sub\u003e and the wurtzite hexagonal phase of CV-ZnO NPs. The SEM images of CV-TiO2 and CV-ZnO nanoparticles revealed their uniform morphology and narrow size distribution (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe slight difference in antioxidant activity between CV-TiO\u003csub\u003e2\u003c/sub\u003e and CV-ZnO nanoparticles could be attributed to their chemical properties and their interaction with the bioactive compounds in the extract (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The findings show that these nanoparticles have a substantial capacity to donate electrons or hydrogen atoms to neutralize free radicals. The presence of phenolic compounds and flavonoids in the bark extract likely contributes to the enhanced antioxidant activity (Xie et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This antioxidant activity is significant in cancer therapy. The slightly higher antioxidant activity induced by CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles compared to CV-ZnO nanoparticles may be due to differences in their catalytic properties and surface chemistry. CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles exhibit photocatalytic activities, which could contribute to their superior performance (Morones et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCV-TiO2 CV-ZnO The antibacterial mechanism of CV-TiO\u003csub\u003e2\u003c/sub\u003e NPs is likely due to the generation of reactive oxygen species (ROS) upon exposure to light. The antibacterial action of CV-ZnO NPs is primarily attributed to the release of Zn\u003csup\u003e2+\u003c/sup\u003e ions, which disrupt bacterial cell membrane integrity, leading to oxidative stress and cell death (Xie et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). NPs may continuously discharge ions which can bind to the cell wall and cytoplasmic membrane due to electrostatic attraction and affinity for sulphur proteins. The attached ions might increase the permeability of the cytoplasmic membrane which causes the bacterial envelope to be disrupted. Because sulphur and phosphorus are key components of DNA, the interaction of ions with these elements can impair DNA replication and cell reproduction. Furthermore, ions can prevent protein synthesis by denaturing ribosomes in the cytoplasm (Morones et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom the results obtained it can be deduced that the concentration of cytotoxicity in the NPs is in concordance with the findings of (Khan et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) which showed toxicity in HCC cells in relation to the concentration of NPs. (Akbari et al., 2011) also provide evidence of mitochondrial impairment involved in cell death since the NPs cause apoptosis of cancer cells through the impairment of caspase pathways. Furthermore, we have also found that green synthesized NPs modulate the cellular redox potential, similar to the recent study by (Agrawal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)that described effects of green NPs on cellular oxidative stress and apoptosis. These comparisons reinforce the significance of our observations within the framework of the nanoparticle-based therapeutic approaches to HCC treatment.The superior antioxidant activity of CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles suggests their potential in combating oxidative stress, a hallmark of HCC (Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Inflammation is a crucial driver of tumorigenesis. Our findings revealed that CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles significantly downregulated the expression of the proinflammatory cytokines in huh-7 HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Numerous studies have demonstrated that alpha-fetoprotein (AFP) suppresses the immune system and is also involved in the MDR process in liver cancer patients. Previous research has shown that when treated with green synthesized nanoparticles (Iqbal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), these inflammatory markers are down-regulated in various human cancer cell lines. PTEN regulates the PI3K/AKT signaling pathway. PTEN loss or mutation can lead to aberrant activation of this pathway and contribute to HCC development (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Zhao et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Bcl-2 inhibits apoptosis. Its expression is abnormally elevated in HCC tissues (Nakopoulou et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Down-regulation of Bcl-2 in HCC cells can inhibit the proliferation and invasion of cancer cells (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bax regulates programmed cell death (Guo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Several studies have shown that Bax expression is reduced in HCC cells, which may confer resistance to apoptosis (Li et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The imbalance between pro-apoptotic and antiapoptotic proteins contributes to tumor cell survival and resistance to therapy. In our study, CV-TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles upregulated the expression of the pro-apoptotic gene Bax in HCC cells.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e nature of our study restricts the translation of our findings to an \u003cem\u003ein vivo\u003c/em\u003e setting. Further studies are warranted to evaluate the efficacy and safety of CV-TiO\u003csub\u003e2\u003c/sub\u003e and CV-ZnO NPs in preclinical and clinical settings. Our study primarily focused on the cytotoxic effects and gene expression changes of green synthesized nanoparticles. It is essential to investigate nanoparticle biodistribution, cellular uptake, and long-term toxicity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current investigation demonstrated the successful synthesis and characterization of CV-TiO\u003csub\u003e2\u003c/sub\u003e and CV-ZnO nanoparticles using \u003cem\u003eCinnamomum verum\u003c/em\u003e bark extract. The characterization techniques, including UV-Vis spectrophotometry, FTIR spectroscopy, XRD analysis, and SEM imaging, confirmed the formation of uniformly dispersed, spherical nanoparticles with high crystallinity and phase purity. The synthesized nanoparticles exhibited significant antioxidant, antibacterial, and antifungal activities. These nanoparticles possessed potent cytotoxic and anti-inflammatory effects against Huh-7 liver cancer cells. Future studies should focus on \u003cem\u003ein vivo\u003c/em\u003e evaluations, long-term safety assessments, and their possible mechanisms of action. The clinical translation of these nanoparticles into therapeutic formulations could pave the way for new and effective treatment regimens for infections and cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R350), King Saud University, Riyadh, Saudi Arabia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Data curation,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Formal analys,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Funding acquisition,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Investigation,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Project administration,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Resources, ; Software, ; Supervision,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Validation, ; Visualization,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Writing – original draft,\u0026nbsp;\u003c/strong\u003e;\u003cstrong\u003e\u0026nbsp;Writing – review \u0026amp; editing,\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAGARWAL, H., KUMAR, S. 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A novel coronavirus from patients with pneumonia in China, 2019. \u003cem\u003eNew England journal of medicine\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eZHU, X., PATHAKOTI, K. \u0026amp; HWANG, H.-M. 2019. Green synthesis of titanium dioxide and zinc oxide nanoparticles and their usage for antimicrobial applications and environmental remediation. \u003cem\u003eGreen synthesis, characterization and applications of nanoparticles.\u003c/em\u003e Elsevier.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Green synthesis, CV-ZnO nanoparticles, CV-TiO2 nanoparticles, Cinnamomum verum, Antibacterial, Antifungal, Anticancer, Nanomedicine","lastPublishedDoi":"10.21203/rs.3.rs-6961371/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6961371/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Green nanotechnology has led the development of novel materials to address the growing concerns of antimicrobial resistance and effective cancer therapies. In the current study, we investigated the potential of biogenic zinc oxide (CV-ZnO ) and titanium dioxide (CV-TiO\u003csub\u003e2 \u003c/sub\u003e) nanoparticles (NPs) synthesized using \u003cem\u003eCinnamomum verum\u003c/em\u003e bark extract to combat human pathogens and assess anticancer potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Green synthesized CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles were characterized using UV-vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), and Scanning Electron Microscopy (SEM). Their antibacterial and antifungal properties were assessed against a panel of pathogenic bacteria and fungi using the disc diffusion method. The antioxidant, cytotoxic, and anti-inflammatory potential was examined against the Huh-7 liver cancer cell line.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The biogenic spherical-shaped CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles exhibited sizes ranging from 40 to 80 nm with absorption peaks at 300-320 nm and 300-400 nm, respectively. FTIR and XRD patterns indicated the presence of hydroxyl and organic groups, confirming high crystallinity, stabilization and phase purity. Both types of NPs exhibited significant antibacterial and antifungal activities, with larger zones of inhibition at higher concentrations. The CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;nanoparticles showed superior antioxidant activity and induced higher levels of superoxide dismutase in Huh-7 cells compared to CV-ZnO \u0026nbsp;nanoparticles. Furthermore, these nanoparticles, especially CV-TiO\u003csub\u003e2\u003c/sub\u003e , exhibited potent cytotoxicity (67.67% at 100 μg/ml) against Huh-7 liver cancer cells in a dose-dependent manner, accompanied by the modulation of key apoptotic (Bax) and inflammatory genes (AFP, Bcl-2, PTEN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe current findings suggest the potential application of biogenic CV-ZnO \u0026nbsp;and CV-TiO\u003csub\u003e2\u003c/sub\u003e \u0026nbsp;NPs in developing novel antimicrobial agents and cancer therapies. Further translational studies are warranted to explore their clinical application.\u003c/p\u003e","manuscriptTitle":"Zinc Oxide and Titanium Dioxide Nanoparticles Bio fabricated for Enhanced Antimicrobic, Antioxidant, and Antitumor Performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 16:19:27","doi":"10.21203/rs.3.rs-6961371/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-14T09:01:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-06T22:36:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288150374714692293150400645457470795403","date":"2025-07-02T06:43:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-28T19:19:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152975722033330224095537763423611508652","date":"2025-06-28T18:03:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98506859916923203068552148444387680555","date":"2025-06-28T13:04:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T05:01:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191978786119708112456166350397754371536","date":"2025-06-27T04:58:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-26T12:43:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-26T12:37:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-26T05:27:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-06-24T04:20:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5fd02cda-7b23-401d-913c-c2bf7402a768","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T16:00:59+00:00","versionOfRecord":{"articleIdentity":"rs-6961371","link":"https://doi.org/10.1007/s12668-025-02154-4","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2025-09-18 15:57:24","publishedOnDateReadable":"September 18th, 2025"},"versionCreatedAt":"2025-06-30 16:19:27","video":"","vorDoi":"10.1007/s12668-025-02154-4","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02154-4","workflowStages":[]},"version":"v1","identity":"rs-6961371","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6961371","identity":"rs-6961371","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}