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INTRODUCTION
Marine macroalgae have gained significance due to their unique biological properties and high nutritional content (Qiu et al., 2022). Among these, G. corticata - red seaweed has been utilized for generations as food and fertilizer, and is a rich source of bioactive compounds like minerals, fatty acids and polyphenols (Chellapandian et al., 2019). This seaweed has recently been explored for green synthesis of nanoparticles (Almeida et al., 2011). The nanoparticles synthesized with G. corticata extract are non- toxic as compared to traditional chemical synthesis and exhibit enhanced functional properties, responsiveness, surface reactivity, and biocompatibility and hence can serve as therapeutic agent (Uzair et al., 2020).
Cancer is a chronic disease affected by oxidative stress due to imbalance between Reactive Oxygen Species (ROS) and defensive mechanisms acting against it (Afzal et al., 2023). The IONPs synthesized using G. corticata extract can serve as therapeutic agent in treating cancer. Therefore, it is necessary to evaluate antioxidant potential to understand their protective function against ROS induced cell damage (Kumar et al., 2021). The cytotoxic potential of IONPs has to be determined to assess safety and effectiveness for therapeutic applications. In vitro cytotoxic model such as A549 lung cancer provide a reliable platform for initial toxicity assessment (Algotiml et al., 2022). This preliminary study investigates G. corticata - mediated IONPs for antioxidant activity and cytotoxic effect on A549 lung cancer cells. This comprehensive strategy emphasizes potential of G. corticata as a sustainable source for developing formulations of biologically active nanoparticles with application in lung cancer treatment.
MATERIALS AND METHODS
Collection of G. corticata
The samples of G. corticata were collected from the Mandapam coast, Rameshwaram, Tamil Nadu, India. The samples were cleaned with distilled water and shade dried for 5-7 days. Samples are powdered and stored for further experimental use (Rosemary et al., 2019).
Nutritional Profiling of G. corticata
The nutritional composition of G. corticata was determined by standard proximate analysis method with simple modifications (Beshaw et al., 2022). All experiments were performed in triplicated.
Estimation of moisture content
The AOAC (2000) method was used to determine moisture content wherein 5 g algal sample was weighed in a pre-weighed crucible and dried in hot air oven at 105ºC until constant weight is achieved and cooled in a desiccator. The samples were reweighed and put back in oven and dried until constant weight achieved. The percent moisture was calculated by equation
w1 - weight of empty crucible,
w2 - weight of crucible+sample before drying,
w3 - Final weight of crucible+sample after drying.
Estimation of Ash Content
2 g of algal sample dried in hot air oven until constant weight achieved. The samples were then transferred to muffle furnace and ashed for 6 hr at 550ºC until white powder is formed. The samples are cooled in desiccator and weighed. Percent ash is calculated by equation (Beshaw et al., 2022).
Estimation of Crude protein Content
Lynch, J. M., & Barbano, D. M. (1999). was employed to estimate protein content. A solution containing 5 g of algal sample, 2 g of Kjeldahl catalyst and 200 mL of sulfuric acid was taken in a digestion flask and boiled until a clear bubble free solution is obtained. Then, 60 mL of water and 6 drops of mixed indicator was added and heated until all nitrogen was distilled. Finally, the obtained solution is titrated against NaOH. Percent crude protein content was calculated using equation.
A - Volume in mL of 0.25N HCl used for titration,
B - Volume in mL of 0.25 N HCl used in Blank titration,
W - Weight of sample in g,
14 - Atomic weight of Nitrogen,
6.25 - Protein - nitrogen conversion factor.
Estimation of Lipid content
To determine lipid content, 5 g of algal sample was taken in Soxhlet and 300 mL of petroleum ether was added and heated 85ºC until solvent completely evaporated. The residues were cooled in a desiccator and reweighed (Beshaw et al., 2022). Percent lipid content was calculated by equation.
w1- weight of empty bottle,
w2 - weight of bottle+Oil,
w3 - weight of sample.
Estimation of Fiber content
4 g of algal sample was mixed in 200 mL of Sulfuric acid and boiled for 30 min and filtered. The residue was washed thrice in hot water and 100 mL of 2% of NaOH was added to mixture and boiled for 30 min. Filtered, washed thrice until free from acid. The residue was dried in hot oven air oven at 100ºC overnight and weighed. Finally, residue was ashed in muffle furnace at 500ºC for 2 hr till grey ash formed and weighed (Beshaw et al., 2022). Percent total crude fiber is calculated by
w1- weight of sample before heating,
w2 - weight of sample after heating,
w3 - weight of original sample.
Estimation of Carbohydrate content
The carbohydrate content calculated by equation (Beshaw et al., 2022).
Green Synthesis of IONPs
Aqueous extract of G. corticata was used for green synthesis of IONPs. 10 g of powdered seaweed was boiled in 100 mL of distilled water for 10 min, cooled, and then filtered through filter paper. The filtrate was mixed with 0.1 M ferric chloride solution in 1:1 ratio. The pH was adjusted to 11, and reaction mixture was continuously stirred for 60 min at room temperature before being allowed to settle for an additional 30 min. The resultant colloidal suspension was centrifuged at 10,000 × g for 15 min. The pellet was washed with ethanol, and dried under vacuum and stored at 4 ºC for futher analysis (Kiwumulo et al., 2022).
Characterization of Nanoparticles
The synthesized IONPs were characterized using UV-visible spectroscopy, scanning across the range of 200 to 800 nm (Rauf et al., 2024), FTIR spectroscopy to identify the functional groups (Minhas et al., 2023), and crystallinity by XRD analysis (Kiwumulo et al., 2022).
Antioxidant Activity
DPPH Antioxidant Assay
2mL of freshly prepared 100mM DPPH in methanol was added to series of concentrations (10,20, 30, 40, and 50 ug/mL) of synthezised IONPs and incubated for 15 mins in dark at 25 C followed by recording absorbance at 517 nm (Jayasree et al., 2018).
ABTS Assay
The ABTS assay was performed with modifications (Sharma and John, 2022). Briefly, ABTS•+ solution was prepared by oxidizing ABTS (7 mM v/v in Milli-Q water) by 2.45 mM Ammonium Persulfate. The mixture was preincubated overnight in dark and diluted in methanol (1:29). Different concengtration of synthesized IONPs were incubated with 2 mL of ABTS•+ reagent at dark for 5 min and the absorbance was measured at 734 nm.
FRAP Assay
The FRAP assay was performed by incubating the synthezised IONPs with 1 mL of FRAP reagent (100 mL of 300 mM Acetate buffer (pH 3.6), 10 mL of 10 mM ferric-tripyridyltriazine (in 40 mM HCl), 10 mL of 20 mM FeCl3, and 12 mL Milli-Q water) for a duration of 10 min, followed by the measurements of absorbance at 593 nm.
Hydrogen Peroxide scavenging Assay
H₂O₂ scavenging activity was determined by incubating IONPs solution with phosphate-buffered H₂O₂ solution (40 mM H₂O₂ in 50 mM phosphate buffer pH 7.4) for 10 min and absorbance measurement at 230 nm (Devi and Kumari, 2022).
Nitric Oxide radical inhibition Assay
For NO scavenging assay, the synthezised IONPs were incubated in 1.5 mL of 10mM sodium nitroprusside in PBS for 30 min and thereafter, addition of 1.5 mL of Griess reagent and immediately absorbance was read at 540 nm (Narasimhan et al., 2013).
Cytotoxicity Studies
The cytotoxic effect of IONPs was evaluated on A549 human lung cancer cell lines. The cells were revived and cultured in DMEM supplemented with 10% FBS and 1% streptomycin. The cells were maintained at 37 ºC with 5% CO₂. Once confluency is achieved, cells were trypsinized and a total of 5 × 10⁴ cells per well were seeded into 96-well plates and cultured overnight. The cells were treated with increasing concentrations (20 - 120 µg/mL) of the synthesized IONPs and incubated for 24 hr. The cells were treated with MTT solution (5 mg/mL), 10 µL into each well and incubated for 4 hr. 100 µL of MTT Dissolving Solution (DMSO) was dispensed into each well to dissolve formazan and absorbance was measured at 570 nm (Narasimhan et al., 2013).
RESULTS
Nutritional Profiling of G. corticata
The proximate composition analysis of G. corticata demonstrated its nutritional profiling as shown in Table 1. A high moisture content of 9.52±2.3% confirms the water-retentive capacity of seaweed, which has implications for storage stability and processing. The ash content was 13.76±1.7%, reflecting a substantial mineral load, a characteristic trait of marine macroalgae. The carbohydrate content was found to be high - 57.40±0.6% indicating rich source of food and fiber content was found to be 3.18±2.7 %, while the protein and lipid contents were 13.39±0.9% and 2.75±1.1%, respectively.
| Sl. No. | Content | Percentage (%) |
|---|---|---|
| 1 | Moisture | 9.52±2.7 |
| 2 | Ash | 13.76±1.7 |
| 3 | Carbohydrate | 57.40±0.6 |
| 4 | Protein | 13.39±0.9 |
| 5 | Lipid | 2.75±1.1 |
| 6 | Ash | 3.18±1.8 |
Synthesis and Characterization of IONPs
The successful green synthesis of IONPs was visually evident by a color shift from yellowish-brown to deep brown, indicating reduction of ferric ions. UV-visible spectroscopy displayed a sharp absorption peak at 294 nm, signifying the surface plasmon resonance typically associated with Fe₃O₄ nanoparticles(Figure 1A). FTIR spectroscopy revealed prominent bands indicative of hydroxyl, carbonyl, and alkene functional groups, confirming the involvement of polysaccharides and phenolics from the seaweed extract in both reduction and capping (Figure 1B). The XRD analysis of green-synthesized iron oxide nanoparticles as shown in Figure 1C revealed broad diffraction peaks at 2θ=31.67° and 33.23°.
Antioxidant Activity
The antioxidant efficacy of G. corticata -mediated IONPs was validated through multiple assays. DPPH radical scavenging (Figure 2A) increased from 63.84% to 87.16% (for 10 µg/mL and 50 µg/mL, respectively). Similar concentration-dependent increase in antioxidant activity was observed in ABTS (67.94% to 87.12%), FRAP (68.53% to 87.11%), H₂O₂ (47.1% to 87.4%), and nitric oxide scavenging (68.21% to 85.62%) assays (Figures 2B-E, respectively). These findings suggest a high radical quenching capacity, which can be attributed to the combined impacts of bioactive compounds of seaweed extract and surface reactivity of IONPs.
Cytotoxicity Studies
The cytotoxic potential of IONPs synthesized from G. corticata was evaluated against A549 lung cancer cells. The results demonstrated a dose-dependent decrease in cell viability following treatment with increasing concentrations of IONPs (20–120 µg/mL) (Figure 3).
Compared with the control group, which showed nearly 100% cell viability, treatment with 20 µg/mL resulted in approximately 86% viability, which gradually decreased to 73% at 40 µg/mL and 59% at 60 µg/mL. A more pronounced cytotoxic effect was observed at higher concentrations, with cell viability reduced to 45% at 80 µg/mL, 22% at 100 µg/mL, and around 10% at 120 µg/mL.
The half-maximal inhibitory concentration (IC₅₀) of the synthesized IONPs against A549 cells was calculated to be 71.347 ± 5.00 µg/mL, indicating a significant inhibitory effect on lung cancer cell proliferation. These findings suggest that G. corticata-mediated IONPs exhibit concentration-dependent cytotoxic activity against A549 lung cancer cells.
DISCUSSION
The proximate analysis of G. corticata show abundant carbohydrates and other nutrient content emphasizing its rich source of macronutrients. This finding corroborates the work of (Rosemary et al., 2019), who emphasized red seaweeds as mineral-rich dietary components.
The IONPs synthesized by G. corticata extract shows no toxicity and are environmentally safe as compared to traditionally synthesized nanoparticles. The IONPs exhibit typical spectral feature consistent with findings by (Anbarasan and Arivalagan, 2024) who reported similar peaks in seaweed-mediated nanoparticle synthesis. FTIR pattern is consistent with the results of (Minhas et al., 2023), where marine polysaccharides facilitated nanoparticle stabilization. The XRD analysis show high amorphous content suggesting partial crystallization influenced by the phytochemicals in the plant extract, consistent with studies of (Kiwumulo et al., 2022).
IONPs exhibit strong antioxidant activity and similar results were reported by (Rajivgandhi et al., 2020), who showed that seaweed-derived nanoparticles enhanced radical scavenging due to their high surface area and synergistic interactions with phenolic constituents. (Long et al., 2022) also highlighted the capacity of brown seaweed-based nanoparticles to eliminate oxidative stress in vivo. Our results reinforce these findings and confirm that red seaweed-based IONPs possess considerable potential in antioxidant therapy and nutraceutical formulations.
The cytotoxic effect of IONPs on A549 lung cancer cells provides a way for development of anti -cancer therapy, though further experimentation is necessary. These findings are consistent with those of (Mohanta et al., 2022), who reported significant cytotoxic effects of seaweed-based iron nanoparticles on various cancer cell lines, attributing their activity to oxidative damage and mitochondrial dysfunction.
CONCLUSION
This study successfully demonstrated the green synthesis of IONPs using the red seaweed G. corticata, highlighting its potential as a sustainable bioresource. Nutritional profiling confirmed the richness of seaweed in essential macronutrients and minerals, supporting its use in functional food and biomedical applications. The synthesized nanoparticles exhibited strong, dose-dependent antioxidant activity across multiple assays and moderate cytotoxicity toward A549 lung cancer cells, indicating promising therapeutic potential. These findings align with existing literature and emphasize the dual role of G. corticata as a candidate in both nutraceuticals and nanomedicine. Future work should focus on detailed mechanistic studies and in vivo evaluations to further explore the clinical relevance and safety of these nanoparticles. Overall, G. corticata-mediated iron oxide nanoparticles present a valuable platform for developing eco-friendly and effective antioxidant, antimicrobial, and anticancer agents.