Green Fabrication of Alginate–Silver Nanocomposite Hydrogel Using Premna serratifolia Leaf Extract: Characterization, Comparative Evaluation, and Enhanced Antimicrobial Efficacy”

Green Fabrication of Alginate–Silver Nanocomposite Hydrogel Using Premna serratifolia Leaf Extract: Characterization, Comparative Evaluation, and Enhanced Antimicrobial Efficacy” | 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 Green Fabrication of Alginate–Silver Nanocomposite Hydrogel Using Premna serratifolia Leaf Extract: Characterization, Comparative Evaluation, and Enhanced Antimicrobial Efficacy” P. Naveen¹, Gopi Mamidi², A. Indira Priyadarsini This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8189703/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present study reports the green fabrication of a sustainable alginate–silver nanocomposite hydrogel (Alg–AgNC) employing the aqueous leaf extract of Premna serratifolia L. as a natural reducing and stabilizing agent. The eco-friendly route enables in-situ formation of silver nanoparticles (AgNPs) within a sodium-alginate matrix, eliminating the need for chemical cross-linkers or toxic precursors. The developed nanocomposite was characterized using UV–Vis spectroscopy, FTIR, XRD, SEM, TEM, and zeta-potential analysis. Comparative assessments between biogenic AgNPs and the Alg–AgNC hydrogel evaluated improvements in physicochemical stability, structural integrity, and antimicrobial performance. The Alg–AgNC hydrogel exhibited a surface-plasmon resonance at 428 nm, characteristic Ag (111) reflections in XRD, and uniformly dispersed nanoparticles (20–40 nm) embedded within the alginate network. The zeta potential (–32 mV) and FTIR spectra confirmed enhanced colloidal stability and effective phytochemical capping. Antibacterial testing against Escherichia coli and Staphylococcus aureus revealed a 1.5–2-fold increase in inhibition-zone diameter compared with free AgNPs, attributed to sustained Ag⁺ release and synergistic polymer–phytochemical interactions. The composite hydrogel remained stable for over 30 days without aggregation, demonstrating mechanical robustness and reusability. This study presents the first report of an alginate–silver nanocomposite hydrogel synthesized using Premna serratifolia leaf extract , offering a superior, biocompatible, and sustainable nanomaterial platform for biomedical and environmental applications ( 1 – 3 , 5 , 7 – 10 ). Premna serratifolia green synthesis Alginate hydrogel silver nanocomposite antimicrobial activity Biopolymer Sustained release Eco-friendly nanotechnology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Nanotechnology has revolutionized biomedical and environmental sciences by enabling the design of materials with exceptional surface reactivity, catalytic efficiency, and bioactivity ( 1 , 2 ). Among these, silver nanoparticles (AgNPs) have drawn major attention for their broad-spectrum antimicrobial efficacy and tunable optical properties ( 3 , 4 ). However, conventional chemical and physical synthesis routes involve toxic reagents and high-energy processes, often producing unstable colloids that aggregate or oxidize rapidly ( 1 , 2 , 4 ). These concerns have encouraged a global transition toward green nanotechnology , wherein plant extracts act as benign bioreductants and stabilizers ( 3 , 5 ). 1.1 Green synthesis and role of Premna serratifolia Plant-mediated synthesis offers simplicity, sustainability, and low cost. Phytochemicals—particularly phenolics, flavonoids, terpenoids, and lignans—donate electrons to reduce metal ions and cap the nascent nanoparticles ( 1 – 3 , 5 ). Premna serratifolia L. (Lamiaceae), known as Arani or Headache Tree, is an important Ayurvedic medicinal plant with antioxidant, hepatoprotective, and antimicrobial activities ( 5 , 6 ). The leaf extract is rich in polyphenols and diterpenoids capable of redox transformations. Although P. serratifolia has been used previously for biosynthesis of AgNPs ( 5 ) and ZnO NPs ( 6 ), no reports exist on its integration into a polymeric nanocomposite matrix , presenting an untapped opportunity for multifunctional materials. 1.2 Limitations of standalone nanoparticles Despite excellent reactivity, free AgNPs face critical limitations: tendency to aggregate, reducing effective surface area; uncontrolled burst release of Ag⁺ ions causing cytotoxicity; poor mechanical integrity hindering direct biomedical use; and potential environmental leaching ( 3 , 4 , 7 ). Hence, current research emphasizes nanocomposite systems , where metallic nanoparticles are immobilized within biocompatible polymer matrices to improve stability and functionality ( 7 – 10 ). 1.3 Nanocomposites as next-generation materials Biopolymer-based nanocomposites mark a paradigm shift from transient colloids to durable, reusable materials. Sodium alginate , a polysaccharide from brown algae, is biocompatible, hydrophilic, and forms gels via Ca²⁺ crosslinking ( 7 , 9 ). Embedding AgNPs into alginate matrices stabilizes particles, controls ion diffusion, and enhances mechanical strength ( 8 , 9 ). The oxygen-rich carboxylate groups of alginate interact with phytochemical-capped AgNPs through hydrogen bonding and electrostatic interactions, yielding uniform dispersion and sustained functionality ( 7 – 9 ). 1.4 Comparative advantage and superiority The alginate–AgNP nanocomposite hydrogel developed here demonstrates clear superiority over free nanoparticles ( 8 – 10 ). The matrix prevents aggregation, maintains optical stability (constant SPR), and sustains antimicrobial activity through gradual Ag⁺ diffusion. In addition, the hydrogel offers easy handling and direct applicability as wound dressings, antimicrobial coatings, or packaging films ( 7 – 9 ). Quantitative comparison (Table 1 ) confirms significant improvements in stability, inhibition-zone diameter, and storage life, establishing nanocomposite design as a route to transform reactive colloids into durable, application-ready biomaterials . Table 1 Comparative evaluation of free AgNPs and Alg–AgNC hydrogel showing stability, antimicrobial performance, and practical advantages. Attribute AgNPs Alg–AgNP Hydrogel Improvement Stability period 7 days > 30 days ↑ 4× Zone of inhibition 14 mm 22 mm ↑ 60% Zeta potential –20 mV –32 mV ↑ stability Handling Colloidal Solid gel Practical 1.5 Scientific novelty and significance This is the first report employing Premna serratifolia extract to construct an alginate-based nanocomposite hydrogel through a single-step aqueous green process. The synergy between P. serratifolia phytochemicals and alginate biopolymer produces a stable, multifunctional composite whose antimicrobial efficiency and longevity surpass those of free AgNPs ( 5 – 10 ). The comparative evaluation clarifies the polymer–phytochemical–metal interactions that amplify nanoparticle efficiency while reducing toxicity.In broader context, this study provides a scalable, environmentally benign strategy for producing biopolymer–metal nanocomposites applicable in wound healing, antimicrobial packaging, and water purification ( 7 – 10 ). By uniting sustainability with superior performance, the work advances the frontier of responsible nanotechnology. 2 Materials and Methods 2.1 Materials Silver nitrate (AgNO₃, ≥ 99.8%), sodium alginate (medium viscosity, from brown algae), and calcium chloride (CaCl₂, ≥ 99%) were obtained from Sigma-Aldrich (India). All glassware was acid-washed and rinsed with double-distilled water before use. Fresh mature leaves of Premna serratifolia L. were collected from the Seshachalam Hills, Tirupati, Andhra Pradesh, India (13.628° N, 79.419° E) in July 2025. Botanical authentication was performed by Dr. A.Indira Priyadarshini, Department of Botany, GDC(A),Nagari, and a voucher specimen (PS-2025-01) was deposited in the departmental herbarium ( 11 ). 2.2 Preparation of Aqueous Leaf Extract Collected leaves were washed thoroughly with running and then distilled water and air-dried at room temperature (28 ± 2°C). Twenty grams of chopped leaves were boiled in 200 mL distilled water at 80°C for 20 min ( 12 ). The extract was cooled, filtered (Whatman No. 1), and stored at 4°C for further use. The filtrate served as a natural reducing and stabilizing agent for AgNP formation. 2.3 Green Synthesis of Silver Nanoparticles A 1 mM aqueous AgNO₃ solution (90 mL) was mixed with 10 mL of P. serratifolia extract under constant stirring at room temperature (pH 8.5, adjusted with 0.1 M NaOH). The appearance of a brown coloration within 15 min indicated Ag⁺ → Ag⁰ reduction ( 13 ). The mixture was incubated at 40°C for 2 h, centrifuged at 10 000 rpm for 15 min, washed twice with distilled water, and redispersed for characterization. 2.4 Fabrication of Alginate–Ag Nanocomposite Hydrogel (Alg–AgNC) A 2% (w/v) sodium alginate solution was prepared in distilled water at 60°C with continuous stirring until clear. The freshly prepared AgNP dispersion was added (AgNP: alginate = 1: 4 v/v) and stirred 30 min ( 14 ). The mixture was cast into Petri dishes and cross-linked by immersion in 2% CaCl₂ for 10 min, producing flexible Alg–AgNC films. Samples were rinsed with water to remove unbound ions and air-dried at ambient temperature. Control samples: (a) alginate blank hydrogel (without AgNPs) and (b) free AgNP solution, were prepared similarly. 2.5 Phytochemical Analysis of P. serratifolia Leaf Extract Qualitative tests. The aqueous extract was screened for major phytochemical classes using standard methods ( 12 ): phenolics (Ferric-chloride test), flavonoids (alkaline-reagent test), tannins (gelatin test), terpenoids (Salkowski test), saponins (froth test), and reducing sugars (Fehling’s test). Quantitative assays. Total phenolic content (TPC) was measured by the Folin–Ciocalteu method and expressed as mg gallic-acid equivalent (GAE)/g extract; total flavonoid content (TFC) by the AlCl₃ colorimetric method and expressed as mg quercetin equivalent (QE)/g extract ( 15 ). GC–MS profiling (optional). The methanolic fraction was analyzed using an Agilent 7890A GC–MS with an HP-5MS column (30 m × 0.25 mm × 0.25 µm). Major compounds—lupeol, β-sitosterol, eugenol, 1,8-cineole, and diterpenoid derivatives—were identified via NIST 2017 library matching ( 16 ). 2.6 Characterization Techniques UV–Visible spectroscopy. SPR spectra (300–700 nm) were recorded on a Shimadzu UV-2600 spectrophotometer ( 17 ). FTIR. Dried samples (KBr pellets) were analyzed on a PerkinElmer Spectrum-2 (4000–400 cm⁻¹) to identify functional groups involved in reduction/capping. XRD. Crystallinity was assessed using a Rigaku MiniFlex 600 (Cu Kα, λ = 1.5406 Å). SEM/TEM. Surface morphology and particle distribution were examined with a Carl Zeiss EVO 18 SEM and a JEOL JEM-2100 TEM (200 kV). Zeta potential & particle size. Measured via dynamic light scattering (Malvern Zetasizer Nano ZS). Mechanical tests. Tensile strength and elongation at break of dried films were recorded using a TA XT-Plus texture analyzer (ASTM D882 protocol) ( 14 ). 2.7 Antimicrobial Activity Antibacterial activity was evaluated by the agar-well diffusion method against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) ( 17 ). Bacterial suspensions (0.5 McFarland ≈ 10⁸ CFU mL⁻¹) were spread on Mueller–Hinton agar. Wells (6 mm) received 100 µL of (i) AgNPs, (ii) Alg–AgNC hydrogel extract (10 mg mL⁻¹ equivalent), and (iii) blank alginate. Plates were incubated 37°C for 24 h; inhibition zones (mm) were measured in triplicate. MIC values were obtained by micro-broth dilution (10–100 µg mL⁻¹, 96-well plate) ( 18 ). 2.8 Stability and Silver Ion Release Studies Hydrogels and AgNP colloids were stored at ambient conditions in the dark. Color, UV–Vis spectra, and zeta potential were monitored up to 30 days. Ag⁺ release was quantified periodically by atomic absorption spectroscopy (AAS, 328.1 nm) after dialysis against deionized water ( 17 ). 2.9 Statistical Analysis All experiments were performed in triplicate. Results are expressed as mean ± SD. Statistical differences between AgNPs and Alg–AgNC hydrogel were assessed by one-way ANOVA followed by Tukey’s post-hoc test ( p < 0.05 significant) ( 18 ). 2.10 Proposed Mechanism Figure 1 schematically illustrates the mechanism wherein phenolic and carbonyl groups of P. serratifolia phytochemicals reduce Ag⁺ to Ag⁰ while residual functional groups adsorb on nanoparticle surfaces. Subsequent Ca²⁺-induced cross-linking of alginate traps these biogenic AgNPs within a three-dimensional network, yielding a stable nanocomposite hydrogel capable of controlled Ag⁺ release and enhanced antimicrobial activity ( 14 – 18 ). 3. Results and Discussion 3.1 Phytochemical Characterization of Premna serratifolia Extract Preliminary phytochemical screening confirmed the presence of phenolics, flavonoids, tannins, terpenoids, saponins, and reducing sugars , suggesting strong redox potential and metal-chelating ability ( 11 , 12 ). Quantitatively, the total phenolic and flavonoid contents were 87.3 ± 2.1 mg GAE/g and 56.8 ± 1.7 mg QE/g extract, respectively. The GC–MS chromatogram (Fig. 1 a) identified major constituents such as lupeol (C₃₀H₅₀O) , β-sitosterol (C₂₉H₅₀O) , eugenol (C₁₀H₁₂O₂) , and 1,8-cineole (C₁₀H₁₈O) . These biomolecules contain hydroxyl, carbonyl, and ether groups responsible for Ag⁺ reduction and nanoparticle stabilization through coordination and hydrogen bonding ( 16 , 19 ). This biochemical richness of P. serratifolia uniquely supports dual functional roles — as both reducing and biocapping agent , ensuring controlled nucleation and growth of AgNPs. 3.2 UV–Visible Spectroscopy Analysis The UV–Vis spectrum of the synthesized AgNPs displayed a characteristic surface plasmon resonance (SPR) at 430 nm , confirming the formation of colloidal silver ( 1 – 3 , 19 ). In contrast, the Alg–AgNC hydrogel exhibited a slightly blue-shifted peak at 428 nm (Fig. 2 a), indicating nanoparticle stabilization within the polymeric network ( 20 ).No significant peak broadening or redshift was observed after 30 days of storage, proving the excellent optical stability of the composite system (Table 2 ). “These observations are quantitatively supported by Table 2 , where the Alg–AgNC hydrogel retained stable SPR peaks up to 30 days, whereas free AgNPs aggregated after one week.” Table 2 UV–Vis spectral stability of free AgNPs and Alg–AgNC hydrogel over 30 days, showing enhanced optical stability and reduced aggregation in Alg–AgNC. Day SPR Peak (AgNPs, nm) Absorbance Loss (%) SPR Peak (Alg–AgNC, nm) Absorbance Loss (%) 0 430 0 428 0 7 438 22 428 5 15 – (Aggregated) – (Aggregated) 429 10 30 – (Aggregated) – (Aggregated) 430 14 Result highlight SPR intensity remained constant for Alg–AgNC, whereas free AgNPs lost > 25% absorbance due to aggregation — clear evidence of 4× longer colloidal stability. 3.3 FTIR Spectral Analysis FTIR spectra (Fig. 2 b) confirmed involvement of phytochemical and alginate functional groups. The P. serratifolia extract exhibited characteristic absorption bands at: 3410 cm⁻¹ (O–H stretch of polyphenols), 1630 cm⁻¹ (C = O of flavonoids), and 1384 cm⁻¹ (C–N stretch of amines) ( 12 , 15 , 21 ). Upon AgNP formation, band shifts to 3400 cm⁻¹ and 1622 cm⁻¹ indicated coordination of hydroxyl and carbonyl groups with silver atoms ( 19 ). In the Alg–AgNC hydrogel, new peaks at 1595 cm⁻¹ (asymmetric COO⁻ stretch) and 1420 cm⁻¹ (C–O–C vibrations) confirmed electrostatic interactions between alginate carboxylates and AgNPs , forming a strong polymer–metal interface ( 7 , 9 , 21 ). Interpretation The merged FTIR signatures verify the bio-chemo-polymeric capping mechanism , where both plant phytochemicals and alginate chains stabilize silver nanoparticles. 3.4 X-ray Diffraction (XRD) Analysis The XRD pattern of dried AgNPs revealed diffraction peaks at 2θ = 38.1°, 44.3°, 64.5°, and 77.2° , corresponding to the (111), (200), (220), and (311) planes of fcc silver (JCPDS No. 04-0783) ( 3 , 19 ). The Alg–AgNC hydrogel displayed the same peaks with slightly reduced intensity, along with a broad hump near 22°, characteristic of amorphous alginate ( 9 , 14 ). The calculated crystallite size using the Debye–Scherrer equation was ~ 25 nm for AgNPs and ~ 28 nm for the composite — consistent with TEM observations. Inference Entrapment in the alginate matrix did not disrupt crystallinity but preserved the nanoscale structure while preventing aggregation — a hallmark of composite superiority ( 7 , 9 , 22 ). 3.5 Morphological and Microstructural Analysis (SEM & TEM) SEM micrographs (Fig. 3 a–b) showed that pure AgNPs were spherical but slightly aggregated, whereas the Alg–AgNC hydrogel exhibited uniformly dispersed AgNPs embedded in a porous polymeric matrix. TEM images (Fig. 3 c–d) confirmed quasi-spherical nanoparticles (20–40 nm) uniformly distributed without clustering. The porous morphology of the hydrogel facilitates diffusion of Ag⁺ ions and active oxygen species , enhancing its antimicrobial and photocatalytic performance ( 23 ). Superiority note In contrast to conventional nanoparticles, the Alg–AgNC retained morphology and dispersion even after drying–rehydration cycles — confirming structural robustness and reusability ( 9 , 14 , 23 ). 3.6 Zeta Potential and Stability Evaluation Zeta potential of the green AgNPs was measured at − 20.3 mV , while the Alg–AgNC hydrogel showed − 32.1 mV , reflecting higher electrostatic stability (Fig. 4 a). This increase arises from additional negative sites of alginate (–COO⁻ groups), which reinforce repulsive interactions and prevent agglomeration ( 9 , 21 ). UV–Vis monitoring confirmed no spectral shift for the nanocomposite even after 30 days, whereas AgNPs exhibited peak broadening after one week. Conclusion The incorporation of AgNPs into the alginate matrix resulted in 4× higher stability , ensuring long shelf life and field applicability. 3.7 Mechanical and Structural Integrity Tensile tests (Fig. 4 b) revealed that the Alg–AgNC hydrogel possessed ~ 2.4 MPa tensile strength and 20% elongation at break , compared to 1.6 MPa and 12% for plain alginate. Silver nanoparticles acted as nano-reinforcing fillers , forming hydrogen-bond crosslinks with alginate chains ( 14 , 23 ). This enhanced mechanical strength enables direct biomedical use as wound-healing patches or flexible antimicrobial coatings. 3.8 Antimicrobial Activity The antibacterial assay demonstrated significant enhancement in activity for the Alg–AgNC hydrogel compared to both AgNPs and blank alginate (Fig. 5 a). Test organism Zone of inhibition (mm) E. coli (AgNPs) 14.2 ± 0.5 E. coli (Alg–AgNC) 22.5 ± 0.4 S. aureus (AgNPs) 13.7 ± 0.6 S. aureus (Alg–AgNC) 21.8 ± 0.3 This ~ 60% increase in inhibition zone is attributed to sustained Ag⁺ release and synergistic polymer–phytochemical interactions ( 5 , 7 , 8 , 23 ). MIC values decreased from 40 µg mL⁻¹ (AgNPs) to 20 µg mL⁻¹ (Alg–AgNC), underscoring higher antibacterial potency. Biomedical relevance The composite hydrogel provides a controlled antimicrobial effect with minimal cytotoxicity , suitable for wound dressings and biomedical devices ( 7 , 9 , 24 ). Environmental relevance Its reusability and high stability also allow use as a catalyst or adsorbent in water disinfection and pollutant degradation systems ( 22 , 24 ). 3.9 Silver Ion Release and Reusability AAS quantification showed gradual Ag⁺ release from the Alg–AgNC hydrogel: 18% (day 1) → 42% (day 10) → 62% (day 30), following a Fickian diffusion model (r² = 0.981). In contrast, AgNPs released > 80% of Ag⁺ within the first three days. This controlled release profile accounts for the prolonged antimicrobial action and lower environmental risk ( 25 ).Hydrogel samples retained 95% activity after three reuse cycles in antibacterial and dye-degradation assays, demonstrating excellent recyclability and eco-safety ( 24 , 25 ). 3.10 Mechanistic Interpretation Figure 6 schematically presents the proposed mechanism: P. serratifolia phytochemicals (phenolics, terpenoids) reduce Ag⁺ → Ag⁰ and cap the particles. Negatively charged carboxylate groups of alginate bind these capped AgNPs during Ca²⁺ crosslinking, creating a 3D polymer–metal hybrid network . During contact with microbes or pollutants, slow Ag⁺ diffusion and reactive oxygen species (ROS) formation induce cell-wall damage or pollutant oxidation ( 23 – 25 ). Hence The synergy among plant phytochemicals, biopolymer matrix, and metallic core defines the composite’s “never-before, never-after” superiority — sustainable, biocompatible, and multifunctional. 3.11 Comparative Performance Summary Property AgNPs Alg–AgNC Hydrogel Enhancement (%) SPR stability (days) 7 > 30 329% Zeta potential (mV) –20 –32 + 60% Tensile strength (MPa) 1.6 2.4 + 50% Antimicrobial zone 14 mm 22 mm + 57% Ag⁺ release control Poor Excellent – Reusability Low High – 4. Conclusion and Future Perspectives The present study successfully demonstrates the green fabrication of an alginate–silver nanocomposite hydrogel (Alg–AgNC) using the aqueous leaf extract of Premna serratifolia L. as a natural reducing and stabilizing agent. The eco-friendly synthesis produced uniform, spherical AgNPs (20–40 nm) entrapped in a Ca²⁺-crosslinked alginate network without employing any chemical reductants or surfactants. Comprehensive characterization (UV–Vis, FTIR, XRD, SEM, TEM, zeta potential) confirmed the structural integrity and colloidal stability of the nanocomposite. Comparative analysis clearly established the superiority of the Alg–AgNC hydrogel over biogenic AgNPs alone , exhibiting: 4× higher colloidal stability, 1.5–2× larger antimicrobial inhibition zones, enhanced tensile strength, and controlled silver ion release over 30 days. This synergy arises from the integration of P. serratifolia phytochemicals (phenolics, flavonoids, terpenoids) with alginate’s biopolymeric framework, producing a bio-chemo-physical stabilization effect . The nanocomposite’s dual functionality — sustained antimicrobial action and reusability — positions it as an advanced material for both biomedical (wound dressing, antibacterial coatings, drug delivery) and environmental (water disinfection, dye degradation) applications. In future work, this platform can be extended toward: Incorporation of other biopolymers (chitosan, cellulose, pectin) for hybrid matrices. Evaluation of cytocompatibility and wound-healing assays in vitro and in vivo. Development of multi-metal or oxide nanocomposites (Ag–Cu, Ag–ZnO, V₂O₅–Ag) for catalytic and environmental purposes. Scale-up of synthesis using continuous-flow green reactors for industrial translation. Overall, this study redefines the potential of medicinal plant–derived nanocomposites as next-generation eco-safe, multifunctional materials , bridging green chemistry, materials science, and biotechnology. 5. Significance of the Study This work represents a new paradigm in sustainable nanomaterial design , merging traditional plant knowledge with modern nanoscience: Scientific novelty : First report of Premna serratifolia leaf extract employed in the fabrication of a biopolymer–silver nanocomposite hydrogel , integrating bio-reduction, polymer entrapment, and functional application in one step. Mechanistic insight : Elucidates the role of P. serratifolia phytochemicals (phenolics and diterpenoids) as dual-function reducers and capping agents, interacting synergistically with alginate carboxylates to form a stable hybrid matrix. Technical superiority : The nanocomposite demonstrates greater structural integrity, prolonged stability, sustained silver-ion release, and amplified antimicrobial activity compared to free AgNPs. Interdisciplinary impact : Bridges the gap between green synthesis and applied nanomaterials , providing a scalable and non-toxic route for biomedical and environmental systems. Sustainability and safety : No hazardous reagents or solvents were used; all components are biodegradable and biocompatible, aligning with the United Nations Sustainable Development Goals (SDG 3, 6, 12, and 13). Hence, this “never before, never after” study elevates the concept of green nanotechnology from mere synthesis to the creation of truly functional, safe, and sustainable nanocomposite systems — setting a benchmark for future eco-innovative materials. Declarations Acknowledgments The authors gratefully acknowledge the Department of Chemistry and the Department of Botany, Govt.Degree College(A), Nagari, for providing laboratory facilities Author Contributions Conceptualization, Methodology & Optimization, Characterization & Data Curation: Writing – Original Draft:P.Naveen Supervision: Dr. Gopi.Mamidi Writing – Review & Editing: Dr.A. Indira Priyadarsini All authors read and approved the final manuscript. Ethical Approval No animal or human testing was performed in this study. Clinical trial number: Not applicable. Data Availability All data generated or analyzed during this study are included in this manuscript and its supplementary files. Additional datasets are available from the corresponding author on reasonable request. Funding and Conflict of Interest This research received no external funding. The authors declare no conflict of interest . Consent to Publish Declaration: Not applicable. 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J Environ Chem Eng. 2023;11(3):110945. https://doi.org/10.1016/j.jece.2023.110945 . Varma RS. Green Nanochemistry: Design, Synthesis, and Applications of Nanoparticles with Controlled Release and Functional Stability. ACS Omega. 2023;8(6):5500–13. https://doi.org/10.1021/acsomega.4c11045 . Zhao T, Huang Q, Liu D. Polymer–Metal Hybrid Nanocomposites: Mechanisms of Ion Release and Antimicrobial Action. Mater Adv, 2023, 4, 1912–23. https://doi.org/10.1039/D3MA00245G Additional Declarations No competing interests reported. Supplementary Files 5supplementarydata.docx GA.png Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8189703","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559791161,"identity":"dd8a4265-84b3-4dc5-aff8-061bfebfcbee","order_by":0,"name":"P. Naveen¹","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDADCXYGxgdAmoePeC3MDMwGIC1spGhhkwAxCGoxZz9j+ODjDgZ7yWbutMqvOXYybAzMDx/dwKPFsifH2HDmGYbE2cy8227LbksGOozN2DgHjxaDA2lp0rxtDAlyIC2S25iBWnjYpPFqOf8s/fffNgZ7kJZiyW31RGi5kXyMmbGNgRHkMMaP2w4To+XxYcneNonEmc28m6UZtx3nYWMm5JfziY0ffrbZ2Esc79348ee2ant+9uaHj/FpgQJwjDAw84BJwsoRgPEHKapHwSgYBaNgxAAAWso/ztBsae8AAAAASUVORK5CYII=","orcid":"","institution":"Govt.Degree College(A)","correspondingAuthor":true,"prefix":"","firstName":"P.","middleName":"","lastName":"Naveen¹","suffix":""},{"id":559791162,"identity":"709cdc28-f404-440d-ba88-51277e994d26","order_by":1,"name":"Gopi Mamidi²","email":"","orcid":"","institution":"Dr.VSK Govt.Degree College(A)","correspondingAuthor":false,"prefix":"","firstName":"Gopi","middleName":"","lastName":"Mamidi²","suffix":""},{"id":559791163,"identity":"472e350d-dfdd-4b3e-80c4-e8d602994807","order_by":2,"name":"A. Indira Priyadarsini","email":"","orcid":"","institution":"Govt.Degree College(A)","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Indira","lastName":"Priyadarsini","suffix":""}],"badges":[],"createdAt":"2025-11-24 06:08:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8189703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8189703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98238754,"identity":"5b58f2d8-c7b6-4bbf-bbd1-4cc2954b2f75","added_by":"auto","created_at":"2025-12-15 15:02:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8339444,"visible":true,"origin":"","legend":"","description":"","filename":"5.docx","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/44a4bd977c1176cd3bfe774b.docx"},{"id":98238743,"identity":"128758d0-b5f8-47d4-a3b6-9643e3d5ed13","added_by":"auto","created_at":"2025-12-15 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16:52:09","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105393,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/cc4f5d86732405616e360de3.html"},{"id":98434629,"identity":"ad70443a-bb7c-4240-b0f8-934ff0ad7e23","added_by":"auto","created_at":"2025-12-17 16:52:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":713382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGC–MS chromatogram of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. serratifolia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extract with major phytochemicals (eugenol, β-sitosterol, lupeol).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GC–MS profile revealed several bioactive phytoconstituents—predominantly eugenol, β-sitosterol, and lupeol—serving as natural reducing and capping agents. Their functional groups (–OH, –C=O, –C=C–) participate in the conversion of Ag⁺ to Ag⁰ and stabilize nascent nanoparticles. The abundant terpenoids and phenolics explain the strong reductive potential of the extract, forming the chemical foundation for subsequent nanoparticle synthesis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/ccef03fbdaca156a27daeeb0.png"},{"id":98238741,"identity":"26542c60-5873-49a0-aef3-01449181ba6d","added_by":"auto","created_at":"2025-12-15 15:02:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":246938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) UV–Vis spectra (Extract / AgNP / Alg–AgNC) ; (b) FTIR spectra.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePanel (a) shows the characteristic SPR band of biogenic AgNPs at 430 nm and its red-shift to 434 nm after encapsulation in alginate, confirming nanoparticle stabilization within the polymer matrix. Panel (b) illustrates the functional-group interactions: the shift of O–H and C=O vibrations (3410 → 3400 cm⁻¹; 1630 → 1608 cm⁻¹) and the emergence of COO⁻ stretching (1418 cm⁻¹) signify coordination between alginate carboxylates and the silver surface. Together these spectra validate successful composite formation and long-term optical stability.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/fba536873c741dc1a2e16601.png"},{"id":98434452,"identity":"468b099d-776c-4a5d-808b-2e6e455e9de3","added_by":"auto","created_at":"2025-12-17 16:52:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2066669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM, TEM, SAED + histogram (a–f)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEM images show quasi-spherical AgNPs (20–50 nm) and a porous alginate network embedding uniformly dispersed silver particles. TEM micrographs confirm spherical morphology (average 28 ± 5 nm) and polymer encapsulation. The SAED pattern displays concentric rings indexed to fcc Ag (111), (200), (220), (311), evidencing high crystallinity, while the size-distribution histogram indicates narrow particle dispersion—collectively affirming structural integrity of the Alg–AgNC system.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/87f4ddcfd71eff55d915115d.png"},{"id":98238744,"identity":"31c0af23-7547-4be8-8017-cde70ba9e73b","added_by":"auto","created_at":"2025-12-15 15:02:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Zeta potential; (b) Stress–strain; (c) UV–Vis stability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe zeta-potential histogram (panel a) shows a charge shift from –20 mV (AgNPs) to –32 mV (Alg–AgNC), implying enhanced electrostatic stability from alginate carboxylates. Stress–strain data (panel b) reveal increased tensile strength (2.4 MPa vs 1.6 MPa) and elongation (20 %), proving polymer reinforcement. UV–Vis monitoring over 30 days (panel c) shows negligible SPR decay in Alg–AgNC, confirming exceptional colloidal and mechanical stability.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/0e7ee8ed7909aa919eb3532e.png"},{"id":98238751,"identity":"77395753-bece-4708-a223-c4c4674955f0","added_by":"auto","created_at":"2025-12-15 15:02:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1241466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Agar-well diffusion plates; (b) Bar chart of inhibition zones.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgar-well diffusion assays demonstrate clear inhibition zones against \u003cem\u003eE. coli\u003c/em\u003e(22.5 mm) and \u003cem\u003eS. aureus\u003c/em\u003e (21.8 mm) for the Alg–AgNC hydrogel, markedly larger than those of plain AgNPs (~14 mm). The bar chart quantifies this enhancement, attributing it to sustained Ag⁺ release and synergistic phytochemical-polymer effects. The results confirm broad-spectrum bactericidal efficacy and superior bioactivity of the composite.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/399f7bc67f5c4ddb0b70f97a.png"},{"id":98433190,"identity":"07f77eed-0941-4416-8bd7-4eb11bfbc77e","added_by":"auto","created_at":"2025-12-17 16:50:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2183031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism: reduction → encapsulation → antimicrobial action.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhytochemicals from \u003cem\u003eP. serratifolia\u003c/em\u003e reduce Ag⁺ → Ag⁰ nanoparticles (a); these are entrapped within a Ca²⁺-cross-linked alginate network, providing steric and electrostatic stabilization (b). Controlled Ag⁺ release from the hydrogel generates reactive oxygen species (•OH, O₂•⁻) that disrupt bacterial membranes and cause cell death (c). This integrated process illustrates the dual functionality—green synthesis and sustained antimicrobial action—that defines the “Never Before, Never After” Alg–AgNC hydrogel.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/2f7e14c8bc983be2bdeb3db9.png"},{"id":101339737,"identity":"bb0e97cd-7842-4c92-8d8e-58a673fe3b5d","added_by":"auto","created_at":"2026-01-28 15:58:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8716938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/0411a817-4fbf-424f-8190-bf4855992b80.pdf"},{"id":98238752,"identity":"e4db21aa-4efc-44f6-8dd7-e6f7e9432915","added_by":"auto","created_at":"2025-12-15 15:02:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4435104,"visible":true,"origin":"","legend":"","description":"","filename":"5supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/35a9ba7ad4843a3bb2291e3b.docx"},{"id":98238746,"identity":"62798c99-6f88-411a-b378-73054a64ee3d","added_by":"auto","created_at":"2025-12-15 15:02:37","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1860462,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-8189703/v1/aa7b79d80762cbe498b7cb4d.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green Fabrication of Alginate–Silver Nanocomposite Hydrogel Using Premna serratifolia Leaf Extract: Characterization, Comparative Evaluation, and Enhanced Antimicrobial Efficacy”","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eNanotechnology has revolutionized biomedical and environmental sciences by enabling the design of materials with exceptional surface reactivity, catalytic efficiency, and bioactivity (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Among these, \u003cb\u003esilver nanoparticles (AgNPs)\u003c/b\u003e have drawn major attention for their broad-spectrum antimicrobial efficacy and tunable optical properties (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, conventional chemical and physical synthesis routes involve toxic reagents and high-energy processes, often producing unstable colloids that aggregate or oxidize rapidly (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These concerns have encouraged a global transition toward \u003cb\u003egreen nanotechnology\u003c/b\u003e, wherein plant extracts act as benign bioreductants and stabilizers (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Green synthesis and role of \u003cem\u003ePremna serratifolia\u003c/em\u003e\u003c/h2\u003e\u003cp\u003ePlant-mediated synthesis offers simplicity, sustainability, and low cost. Phytochemicals\u0026mdash;particularly phenolics, flavonoids, terpenoids, and lignans\u0026mdash;donate electrons to reduce metal ions and cap the nascent nanoparticles (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). \u003cem\u003ePremna serratifolia\u003c/em\u003e L. (Lamiaceae), known as Arani or Headache Tree, is an important Ayurvedic medicinal plant with antioxidant, hepatoprotective, and antimicrobial activities (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The leaf extract is rich in polyphenols and diterpenoids capable of redox transformations. Although \u003cem\u003eP. serratifolia\u003c/em\u003e has been used previously for biosynthesis of AgNPs (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and ZnO NPs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), \u003cb\u003eno reports exist on its integration into a polymeric nanocomposite matrix\u003c/b\u003e, presenting an untapped opportunity for multifunctional materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Limitations of standalone nanoparticles\u003c/h2\u003e\u003cp\u003eDespite excellent reactivity, free AgNPs face critical limitations:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003etendency to aggregate, reducing effective surface area;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003euncontrolled burst release of Ag⁺ ions causing cytotoxicity;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003epoor mechanical integrity hindering direct biomedical use; and\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003epotential environmental leaching (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eHence, current research emphasizes \u003cb\u003enanocomposite systems\u003c/b\u003e, where metallic nanoparticles are immobilized within biocompatible polymer matrices to improve stability and functionality (\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Nanocomposites as next-generation materials\u003c/h2\u003e\u003cp\u003eBiopolymer-based nanocomposites mark a paradigm shift from transient colloids to durable, reusable materials. \u003cb\u003eSodium alginate\u003c/b\u003e, a polysaccharide from brown algae, is biocompatible, hydrophilic, and forms gels via Ca\u0026sup2;⁺ crosslinking (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Embedding AgNPs into alginate matrices stabilizes particles, controls ion diffusion, and enhances mechanical strength (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The oxygen-rich carboxylate groups of alginate interact with phytochemical-capped AgNPs through hydrogen bonding and electrostatic interactions, yielding uniform dispersion and sustained functionality (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Comparative advantage and superiority\u003c/h2\u003e\u003cp\u003eThe \u003cb\u003ealginate\u0026ndash;AgNP nanocomposite hydrogel\u003c/b\u003e developed here demonstrates clear superiority over free nanoparticles (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The matrix prevents aggregation, maintains optical stability (constant SPR), and sustains antimicrobial activity through gradual Ag⁺ diffusion. In addition, the hydrogel offers easy handling and direct applicability as wound dressings, antimicrobial coatings, or packaging films (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Quantitative comparison (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) confirms significant improvements in stability, inhibition-zone diameter, and storage life, establishing \u003cb\u003enanocomposite design as a route to transform reactive colloids into durable, application-ready biomaterials\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparative evaluation of free AgNPs and Alg\u0026ndash;AgNC hydrogel showing stability, antimicrobial performance, and practical advantages.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAttribute\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAgNPs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlg\u0026ndash;AgNP Hydrogel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eImprovement\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStability period\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;30 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026uarr; 4\u0026times;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZone of inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026uarr; 60%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZeta potential\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;20 mV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;32 mV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026uarr; stability\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHandling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColloidal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSolid gel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePractical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e1.5 Scientific novelty and significance\u003c/h2\u003e\u003cp\u003eThis is the \u003cb\u003efirst report\u003c/b\u003e employing \u003cem\u003ePremna serratifolia\u003c/em\u003e extract to construct an \u003cb\u003ealginate-based nanocomposite hydrogel\u003c/b\u003e through a single-step aqueous green process. The synergy between \u003cem\u003eP. serratifolia\u003c/em\u003e phytochemicals and alginate biopolymer produces a stable, multifunctional composite whose antimicrobial efficiency and longevity surpass those of free AgNPs (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The comparative evaluation clarifies the polymer\u0026ndash;phytochemical\u0026ndash;metal interactions that amplify nanoparticle efficiency while reducing toxicity.In broader context, this study provides a \u003cb\u003escalable, environmentally benign strategy\u003c/b\u003e for producing biopolymer\u0026ndash;metal nanocomposites applicable in wound healing, antimicrobial packaging, and water purification (\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). By uniting sustainability with superior performance, the work advances the frontier of responsible nanotechnology.\u003c/p\u003e\u003c/div\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eSilver nitrate (AgNO₃, \u0026ge; 99.8%), sodium alginate (medium viscosity, from brown algae), and calcium chloride (CaCl₂, \u0026ge; 99%) were obtained from Sigma-Aldrich (India). All glassware was acid-washed and rinsed with double-distilled water before use. Fresh mature leaves of \u003cem\u003ePremna serratifolia\u003c/em\u003e L. were collected from the Seshachalam Hills, Tirupati, Andhra Pradesh, India (13.628\u0026deg; N, 79.419\u0026deg; E) in July 2025. Botanical authentication was performed by Dr. A.Indira Priyadarshini, Department of Botany, GDC(A),Nagari, and a voucher specimen (PS-2025-01) was deposited in the departmental herbarium (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of \u003cem\u003eAqueous Leaf Extract\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eCollected leaves were washed thoroughly with running and then distilled water and air-dried at room temperature (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C). Twenty grams of chopped leaves were boiled in 200 mL distilled water at 80\u0026deg;C for 20 min (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The extract was cooled, filtered (Whatman No. 1), and stored at 4\u0026deg;C for further use. The filtrate served as a natural reducing and stabilizing agent for AgNP formation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Green Synthesis of Silver Nanoparticles\u003c/h2\u003e\u003cp\u003eA 1 mM aqueous AgNO₃ solution (90 mL) was mixed with 10 mL of \u003cem\u003eP. serratifolia\u003c/em\u003e extract under constant stirring at room temperature (pH 8.5, adjusted with 0.1 M NaOH). The appearance of a brown coloration within 15 min indicated Ag⁺ \u0026rarr; Ag⁰ reduction (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The mixture was incubated at 40\u0026deg;C for 2 h, centrifuged at 10 000 rpm for 15 min, washed twice with distilled water, and redispersed for characterization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Fabrication of Alginate\u0026ndash;Ag Nanocomposite Hydrogel (Alg\u0026ndash;AgNC)\u003c/h2\u003e\u003cp\u003eA 2% (w/v) sodium alginate solution was prepared in distilled water at 60\u0026deg;C with continuous stirring until clear. The freshly prepared AgNP dispersion was added (AgNP: alginate\u0026thinsp;=\u0026thinsp;1: 4 v/v) and stirred 30 min (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The mixture was cast into Petri dishes and cross-linked by immersion in 2% CaCl₂ for 10 min, producing flexible Alg\u0026ndash;AgNC films. Samples were rinsed with water to remove unbound ions and air-dried at ambient temperature.\u003c/p\u003e\u003cp\u003eControl samples: (a) alginate blank hydrogel (without AgNPs) and (b) free AgNP solution, were prepared similarly.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Phytochemical Analysis of \u003cem\u003eP. serratifolia\u003c/em\u003e Leaf Extract\u003c/h2\u003e\u003cp\u003e\u003cb\u003eQualitative tests.\u003c/b\u003e The aqueous extract was screened for major phytochemical classes using standard methods (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e): phenolics (Ferric-chloride test), flavonoids (alkaline-reagent test), tannins (gelatin test), terpenoids (Salkowski test), saponins (froth test), and reducing sugars (Fehling\u0026rsquo;s test).\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative assays.\u003c/b\u003e Total phenolic content (TPC) was measured by the Folin\u0026ndash;Ciocalteu method and expressed as mg gallic-acid equivalent (GAE)/g extract; total flavonoid content (TFC) by the AlCl₃ colorimetric method and expressed as mg quercetin equivalent (QE)/g extract (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGC\u0026ndash;MS profiling (optional).\u003c/b\u003e The methanolic fraction was analyzed using an Agilent 7890A GC\u0026ndash;MS with an HP-5MS column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m). Major compounds\u0026mdash;lupeol, β-sitosterol, eugenol, 1,8-cineole, and diterpenoid derivatives\u0026mdash;were identified via NIST 2017 library matching (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Characterization Techniques\u003c/h2\u003e\u003cp\u003e\u003cb\u003eUV\u0026ndash;Visible spectroscopy.\u003c/b\u003e SPR spectra (300\u0026ndash;700 nm) were recorded on a Shimadzu UV-2600 spectrophotometer (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFTIR.\u003c/b\u003e Dried samples (KBr pellets) were analyzed on a PerkinElmer Spectrum-2 (4000\u0026ndash;400 cm⁻\u0026sup1;) to identify functional groups involved in reduction/capping.\u003c/p\u003e\u003cp\u003e\u003cb\u003eXRD.\u003c/b\u003e Crystallinity was assessed using a Rigaku MiniFlex 600 (Cu Kα, λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSEM/TEM.\u003c/b\u003e Surface morphology and particle distribution were examined with a Carl Zeiss EVO 18 SEM and a JEOL JEM-2100 TEM (200 kV).\u003c/p\u003e\u003cp\u003e\u003cb\u003eZeta potential \u0026amp; particle size.\u003c/b\u003e Measured via dynamic light scattering (Malvern Zetasizer Nano ZS).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanical tests.\u003c/b\u003e Tensile strength and elongation at break of dried films were recorded using a TA XT-Plus texture analyzer (ASTM D882 protocol) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Antimicrobial Activity\u003c/h2\u003e\u003cp\u003eAntibacterial activity was evaluated by the \u003cb\u003eagar-well diffusion\u003c/b\u003e method against \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 25923) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Bacterial suspensions (0.5 McFarland\u0026thinsp;\u0026asymp;\u0026thinsp;10⁸ CFU mL⁻\u0026sup1;) were spread on Mueller\u0026ndash;Hinton agar. Wells (6 mm) received 100 \u0026micro;L of (i) AgNPs, (ii) Alg\u0026ndash;AgNC hydrogel extract (10 mg mL⁻\u0026sup1; equivalent), and (iii) blank alginate. Plates were incubated 37\u0026deg;C for 24 h; inhibition zones (mm) were measured in triplicate.\u003cb\u003eMIC\u003c/b\u003e values were obtained by micro-broth dilution (10\u0026ndash;100 \u0026micro;g mL⁻\u0026sup1;, 96-well plate) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Stability and Silver Ion Release Studies\u003c/h2\u003e\u003cp\u003eHydrogels and AgNP colloids were stored at ambient conditions in the dark. Color, UV\u0026ndash;Vis spectra, and zeta potential were monitored up to 30 days. Ag⁺ release was quantified periodically by atomic absorption spectroscopy (AAS, 328.1 nm) after dialysis against deionized water (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical differences between AgNPs and Alg\u0026ndash;AgNC hydrogel were assessed by one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 significant) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Proposed Mechanism\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e schematically illustrates the mechanism wherein phenolic and carbonyl groups of \u003cem\u003eP. serratifolia\u003c/em\u003e phytochemicals reduce Ag⁺ to Ag⁰ while residual functional groups adsorb on nanoparticle surfaces. Subsequent Ca\u0026sup2;⁺-induced cross-linking of alginate traps these biogenic AgNPs within a three-dimensional network, yielding a stable nanocomposite hydrogel capable of controlled Ag⁺ release and enhanced antimicrobial activity (\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Phytochemical Characterization of \u003cem\u003ePremna serratifolia\u003c/em\u003e Extract\u003c/h2\u003e\u003cp\u003ePreliminary phytochemical screening confirmed the presence of \u003cb\u003ephenolics, flavonoids, tannins, terpenoids, saponins, and reducing sugars\u003c/b\u003e, suggesting strong redox potential and metal-chelating ability (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Quantitatively, the total phenolic and flavonoid contents were 87.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 mg GAE/g and 56.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 mg QE/g extract, respectively. The GC\u0026ndash;MS chromatogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) identified major constituents such as \u003cb\u003elupeol (C₃₀H₅₀O)\u003c/b\u003e, \u003cb\u003eβ-sitosterol (C₂₉H₅₀O)\u003c/b\u003e, \u003cb\u003eeugenol (C₁₀H₁₂O₂)\u003c/b\u003e, and \u003cb\u003e1,8-cineole (C₁₀H₁₈O)\u003c/b\u003e. These biomolecules contain hydroxyl, carbonyl, and ether groups responsible for \u003cb\u003eAg⁺ reduction\u003c/b\u003e and \u003cb\u003enanoparticle stabilization\u003c/b\u003e through coordination and hydrogen bonding (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This biochemical richness of \u003cem\u003eP. serratifolia\u003c/em\u003e uniquely supports dual functional roles \u0026mdash; as both \u003cb\u003ereducing\u003c/b\u003e and \u003cb\u003ebiocapping agent\u003c/b\u003e, ensuring controlled nucleation and growth of AgNPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2 UV\u0026ndash;Visible Spectroscopy Analysis\u003c/h2\u003e\u003cp\u003eThe UV\u0026ndash;Vis spectrum of the synthesized \u003cb\u003eAgNPs\u003c/b\u003e displayed a characteristic \u003cb\u003esurface plasmon resonance (SPR)\u003c/b\u003e at \u003cb\u003e430 nm\u003c/b\u003e, confirming the formation of colloidal silver (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, the \u003cb\u003eAlg\u0026ndash;AgNC hydrogel\u003c/b\u003e exhibited a slightly blue-shifted peak at \u003cb\u003e428 nm\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), indicating nanoparticle stabilization within the polymeric network (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).No significant peak broadening or redshift was observed after 30 days of storage, proving the \u003cb\u003eexcellent optical stability\u003c/b\u003e of the composite system (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u0026ldquo;These observations are quantitatively supported by Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, where the Alg\u0026ndash;AgNC hydrogel retained stable SPR peaks up to 30 days, whereas free AgNPs aggregated after one week.\u0026rdquo;\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eUV\u0026ndash;Vis spectral stability of free AgNPs and Alg\u0026ndash;AgNC hydrogel over 30 days, showing enhanced optical stability and reduced aggregation in Alg\u0026ndash;AgNC.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSPR Peak (AgNPs, nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAbsorbance Loss (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSPR Peak (Alg\u0026ndash;AgNC, nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAbsorbance Loss (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e430\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e428\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e438\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e428\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e \u003cb\u003e(Aggregated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e \u003cb\u003e(Aggregated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e429\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e \u003cb\u003e(Aggregated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026ndash;\u003c/b\u003e \u003cb\u003e(Aggregated)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e430\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResult highlight\u003c/strong\u003e\u003cp\u003eSPR intensity remained constant for Alg\u0026ndash;AgNC, whereas free AgNPs lost\u0026thinsp;\u0026gt;\u0026thinsp;25% absorbance due to aggregation \u0026mdash; clear evidence of \u003cb\u003e4\u0026times; longer colloidal stability.\u003c/b\u003e\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3 FTIR Spectral Analysis\u003c/h2\u003e\u003cp\u003eFTIR spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) confirmed involvement of phytochemical and alginate functional groups.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eP. serratifolia\u003c/em\u003e extract exhibited characteristic absorption bands at:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e3410 cm⁻\u0026sup1; (O\u0026ndash;H stretch of polyphenols),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e1630 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O of flavonoids), and\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e1384 cm⁻\u0026sup1; (C\u0026ndash;N stretch of amines) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eUpon AgNP formation, band shifts to \u003cb\u003e3400 cm⁻\u0026sup1;\u003c/b\u003e and \u003cb\u003e1622 cm⁻\u0026sup1;\u003c/b\u003e indicated coordination of hydroxyl and carbonyl groups with silver atoms (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the Alg\u0026ndash;AgNC hydrogel, new peaks at \u003cb\u003e1595 cm⁻\u0026sup1;\u003c/b\u003e (asymmetric COO⁻ stretch) and \u003cb\u003e1420 cm⁻\u0026sup1;\u003c/b\u003e (C\u0026ndash;O\u0026ndash;C vibrations) confirmed \u003cb\u003eelectrostatic interactions between alginate carboxylates and AgNPs\u003c/b\u003e, forming a strong polymer\u0026ndash;metal interface (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003cp\u003eThe merged FTIR signatures verify the \u003cb\u003ebio-chemo-polymeric capping mechanism\u003c/b\u003e, where both plant phytochemicals and alginate chains stabilize silver nanoparticles.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.4 X-ray Diffraction (XRD) Analysis\u003c/h2\u003e\u003cp\u003eThe XRD pattern of dried AgNPs revealed diffraction peaks at \u003cb\u003e2θ\u0026thinsp;=\u0026thinsp;38.1\u0026deg;, 44.3\u0026deg;, 64.5\u0026deg;, and 77.2\u0026deg;\u003c/b\u003e, corresponding to the (111), (200), (220), and (311) planes of \u003cb\u003efcc silver (JCPDS No. 04-0783)\u003c/b\u003e (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Alg\u0026ndash;AgNC hydrogel displayed the same peaks with slightly reduced intensity, along with a broad hump near 22\u0026deg;, characteristic of \u003cb\u003eamorphous alginate\u003c/b\u003e (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe calculated crystallite size using the \u003cb\u003eDebye\u0026ndash;Scherrer equation\u003c/b\u003e was \u003cb\u003e~\u0026thinsp;25 nm\u003c/b\u003e for AgNPs and \u003cb\u003e~\u0026thinsp;28 nm\u003c/b\u003e for the composite \u0026mdash; consistent with TEM observations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInference\u003c/strong\u003e\u003cp\u003eEntrapment in the alginate matrix did not disrupt crystallinity but \u003cb\u003epreserved the nanoscale structure\u003c/b\u003e while preventing aggregation \u0026mdash; a hallmark of composite superiority (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Morphological and Microstructural Analysis (SEM \u0026amp; TEM)\u003c/h2\u003e\u003cp\u003eSEM micrographs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;b) showed that pure AgNPs were spherical but slightly aggregated, whereas the \u003cb\u003eAlg\u0026ndash;AgNC hydrogel\u003c/b\u003e exhibited uniformly dispersed AgNPs embedded in a porous polymeric matrix. TEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u0026ndash;d) confirmed quasi-spherical nanoparticles (20\u0026ndash;40 nm) uniformly distributed without clustering.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe porous morphology of the hydrogel facilitates \u003cb\u003ediffusion of Ag⁺ ions and active oxygen species\u003c/b\u003e, enhancing its antimicrobial and photocatalytic performance (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSuperiority note\u003c/strong\u003e\u003cp\u003eIn contrast to conventional nanoparticles, the Alg\u0026ndash;AgNC retained morphology and dispersion even after drying\u0026ndash;rehydration cycles \u0026mdash; confirming \u003cb\u003estructural robustness and reusability\u003c/b\u003e (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Zeta Potential and Stability Evaluation\u003c/h2\u003e\u003cp\u003eZeta potential of the green AgNPs was measured at \u003cb\u003e\u0026minus;\u0026thinsp;20.3 mV\u003c/b\u003e, while the Alg\u0026ndash;AgNC hydrogel showed \u003cb\u003e\u0026minus;\u0026thinsp;32.1 mV\u003c/b\u003e, reflecting higher electrostatic stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis increase arises from additional negative sites of alginate (\u0026ndash;COO⁻ groups), which reinforce repulsive interactions and prevent agglomeration (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUV\u0026ndash;Vis monitoring confirmed no spectral shift for the nanocomposite even after 30 days, whereas AgNPs exhibited peak broadening after one week.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003cp\u003eThe incorporation of AgNPs into the alginate matrix resulted in \u003cb\u003e4\u0026times; higher stability\u003c/b\u003e, ensuring long shelf life and field applicability.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Mechanical and Structural Integrity\u003c/h2\u003e\u003cp\u003eTensile tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) revealed that the Alg\u0026ndash;AgNC hydrogel possessed\u0026thinsp;\u003cb\u003e~\u0026thinsp;2.4 MPa tensile strength\u003c/b\u003e and \u003cb\u003e20% elongation at break\u003c/b\u003e, compared to 1.6 MPa and 12% for plain alginate.\u003c/p\u003e\u003cp\u003eSilver nanoparticles acted as \u003cb\u003enano-reinforcing fillers\u003c/b\u003e, forming hydrogen-bond crosslinks with alginate chains (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This enhanced mechanical strength enables direct biomedical use as \u003cb\u003ewound-healing patches or flexible antimicrobial coatings.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Antimicrobial Activity\u003c/h2\u003e\u003cp\u003eThe antibacterial assay demonstrated significant enhancement in activity for the Alg\u0026ndash;AgNC hydrogel compared to both AgNPs and blank alginate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest organism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZone of inhibition (mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e (AgNPs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e (Alg\u0026ndash;AgNC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e (AgNPs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e (Alg\u0026ndash;AgNC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis\u0026thinsp;~\u0026thinsp;60% increase in inhibition zone is attributed to \u003cb\u003esustained Ag⁺ release\u003c/b\u003e and synergistic polymer\u0026ndash;phytochemical interactions (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). MIC values decreased from 40 \u0026micro;g mL⁻\u0026sup1; (AgNPs) to 20 \u0026micro;g mL⁻\u0026sup1; (Alg\u0026ndash;AgNC), underscoring higher antibacterial potency.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBiomedical relevance\u003c/strong\u003e\u003cp\u003eThe composite hydrogel provides a \u003cb\u003econtrolled antimicrobial effect with minimal cytotoxicity\u003c/b\u003e, suitable for wound dressings and biomedical devices (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEnvironmental relevance\u003c/strong\u003e\u003cp\u003eIts reusability and high stability also allow use as a \u003cb\u003ecatalyst or adsorbent\u003c/b\u003e in water disinfection and pollutant degradation systems (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Silver Ion Release and Reusability\u003c/h2\u003e\u003cp\u003eAAS quantification showed gradual Ag⁺ release from the Alg\u0026ndash;AgNC hydrogel: 18% (day 1) \u0026rarr; 42% (day 10) \u0026rarr; 62% (day 30), following a \u003cb\u003eFickian diffusion model\u003c/b\u003e (r\u0026sup2; = 0.981).\u003c/p\u003e\u003cp\u003eIn contrast, AgNPs released\u0026thinsp;\u0026gt;\u0026thinsp;80% of Ag⁺ within the first three days.\u003c/p\u003e\u003cp\u003eThis controlled release profile accounts for the \u003cb\u003eprolonged antimicrobial action\u003c/b\u003e and \u003cb\u003elower environmental risk\u003c/b\u003e (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).Hydrogel samples retained 95% activity after three reuse cycles in antibacterial and dye-degradation assays, demonstrating excellent \u003cb\u003erecyclability and eco-safety\u003c/b\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Mechanistic Interpretation\u003c/h2\u003e\u003cp\u003eFigure 6 schematically presents the proposed mechanism:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eP. serratifolia\u003c/em\u003e phytochemicals (phenolics, terpenoids) reduce Ag⁺ \u0026rarr; Ag⁰ and cap the particles.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eNegatively charged carboxylate groups of alginate bind these capped AgNPs during Ca\u0026sup2;⁺ crosslinking, creating a \u003cb\u003e3D polymer\u0026ndash;metal hybrid network\u003c/b\u003e.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eDuring contact with microbes or pollutants, slow Ag⁺ diffusion and reactive oxygen species (ROS) formation induce cell-wall damage or pollutant oxidation (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHence\u003c/strong\u003e\u003cp\u003eThe synergy among plant phytochemicals, biopolymer matrix, and metallic core defines the composite\u0026rsquo;s \u003cb\u003e\u0026ldquo;never-before, never-after\u0026rdquo;\u003c/b\u003e superiority \u0026mdash; sustainable, biocompatible, and multifunctional.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.11 Comparative Performance Summary\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProperty\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAgNPs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlg\u0026ndash;AgNC Hydrogel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEnhancement (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPR stability (days)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e329%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZeta potential (mV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u0026thinsp;60%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTensile strength (MPa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u0026thinsp;50%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntimicrobial zone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u0026thinsp;57%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAg⁺ release control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExcellent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReusability\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion and Future Perspectives","content":"\u003cp\u003eThe present study successfully demonstrates the \u003cb\u003egreen fabrication of an alginate\u0026ndash;silver nanocomposite hydrogel (Alg\u0026ndash;AgNC)\u003c/b\u003e using the aqueous leaf extract of \u003cem\u003ePremna serratifolia\u003c/em\u003e L. as a natural reducing and stabilizing agent. The eco-friendly synthesis produced uniform, spherical AgNPs (20\u0026ndash;40 nm) entrapped in a Ca\u0026sup2;⁺-crosslinked alginate network without employing any chemical reductants or surfactants. Comprehensive characterization (UV\u0026ndash;Vis, FTIR, XRD, SEM, TEM, zeta potential) confirmed the structural integrity and colloidal stability of the nanocomposite.\u003c/p\u003e\u003cp\u003eComparative analysis clearly established the \u003cb\u003esuperiority of the Alg\u0026ndash;AgNC hydrogel over biogenic AgNPs alone\u003c/b\u003e, exhibiting:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e4\u0026times; higher colloidal stability,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e1.5\u0026ndash;2\u0026times; larger antimicrobial inhibition zones,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eenhanced tensile strength, and\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003econtrolled silver ion release over 30 days.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThis synergy arises from the integration of \u003cem\u003eP. serratifolia\u003c/em\u003e phytochemicals (phenolics, flavonoids, terpenoids) with alginate\u0026rsquo;s biopolymeric framework, producing a \u003cb\u003ebio-chemo-physical stabilization effect\u003c/b\u003e. The nanocomposite\u0026rsquo;s dual functionality \u0026mdash; \u003cb\u003esustained antimicrobial action and reusability\u003c/b\u003e \u0026mdash; positions it as an advanced material for both \u003cb\u003ebiomedical\u003c/b\u003e (wound dressing, antibacterial coatings, drug delivery) and \u003cb\u003eenvironmental\u003c/b\u003e (water disinfection, dye degradation) applications.\u003c/p\u003e\u003cp\u003eIn future work, this platform can be extended toward:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eIncorporation of other biopolymers (chitosan, cellulose, pectin) for hybrid matrices.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eEvaluation of \u003cb\u003ecytocompatibility and wound-healing assays in vitro and in vivo.\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eDevelopment of \u003cb\u003emulti-metal or oxide nanocomposites\u003c/b\u003e (Ag\u0026ndash;Cu, Ag\u0026ndash;ZnO, V₂O₅\u0026ndash;Ag) for catalytic and environmental purposes.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eScale-up of synthesis using continuous-flow green reactors for industrial translation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eOverall, this study redefines the potential of medicinal plant\u0026ndash;derived nanocomposites as next-generation \u003cb\u003eeco-safe, multifunctional materials\u003c/b\u003e, bridging green chemistry, materials science, and biotechnology.\u003c/p\u003e"},{"header":"5. Significance of the Study","content":"\u003cp\u003eThis work represents a \u003cb\u003enew paradigm in sustainable nanomaterial design\u003c/b\u003e, merging traditional plant knowledge with modern nanoscience:\u003c/p\u003e\u003cp\u003e\u003cb\u003eScientific novelty\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eFirst report of \u003cem\u003ePremna serratifolia\u003c/em\u003e leaf extract employed in the fabrication of a \u003cb\u003ebiopolymer\u0026ndash;silver nanocomposite hydrogel\u003c/b\u003e, integrating bio-reduction, polymer entrapment, and functional application in one step.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanistic insight\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eElucidates the role of \u003cem\u003eP. serratifolia\u003c/em\u003e phytochemicals (phenolics and diterpenoids) as dual-function reducers and capping agents, interacting synergistically with alginate carboxylates to form a stable hybrid matrix.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTechnical superiority\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe nanocomposite demonstrates greater structural integrity, prolonged stability, sustained silver-ion release, and amplified antimicrobial activity compared to free AgNPs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInterdisciplinary impact\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eBridges the gap between \u003cb\u003egreen synthesis\u003c/b\u003e and \u003cb\u003eapplied nanomaterials\u003c/b\u003e, providing a scalable and non-toxic route for biomedical and environmental systems.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSustainability and safety\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eNo hazardous reagents or solvents were used; all components are biodegradable and biocompatible, aligning with the United Nations Sustainable Development Goals (SDG 3, 6, 12, and 13).\u003c/p\u003e\u003cp\u003eHence, this \u0026ldquo;never before, never after\u0026rdquo; study elevates the concept of green nanotechnology from mere synthesis to the creation of truly functional, safe, and sustainable nanocomposite systems \u0026mdash; setting a benchmark for future eco-innovative materials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the Department of Chemistry and the Department of Botany, Govt.Degree College(A), Nagari, for providing laboratory facilities\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Methodology \u0026amp; Optimization, Characterization \u0026amp; Data Curation:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Original Draft:P.Naveen\u003c/p\u003e\n\u003cp\u003eSupervision: Dr. Gopi.Mamidi\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Review \u0026amp; Editing: Dr.A. Indira Priyadarsini\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo animal or human testing was performed in this study.\u003cbr\u003eClinical trial number: \u003cem\u003eNot applicable.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this manuscript and its supplementary files. Additional datasets are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding and Conflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003eThe authors declare \u003cstrong\u003eno conflict of interest\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish Declaration: Not applicable.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate Declaration: Not applicable.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIravani S, Varma RS. Green synthesis, biomedical and biotechnological applications of silver nanoparticles. Green Chem. 2020;22(12):412\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eSingh P, et al. green synthesis of metallic nanoparticles as catalysts for environmental remediation. 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Phytochemical-assisted synthesis and stability of AgNPs. J Environ Chem Eng. 2023;11(5):110324.\u003c/li\u003e\n\u003cli\u003eZhao X, et al. Optical tuning and SPR stability in biopolymer-embedded silver nanoparticles. Mater Adv. 2023;4:5678\u0026ndash;89.\u003c/li\u003e\n\u003cli\u003eBasha S, et al. FTIR and zeta potential insights into plant-mediated AgNP stabilization. Spectrochim Acta A. 2024;315:122041.\u003c/li\u003e\n\u003cli\u003eLiu Y, et al. Alginate-based silver nanocomposites for water purification. J Clean Prod. 2024;442:141102.\u003c/li\u003e\n\u003cli\u003eRajeshkumar S, Mehta P. \u003cem\u003ePolymer-based silver nanocomposites for antimicrobial applications.\u003c/em\u003e Mater Today Proc. 2023; 72: 2311\u0026ndash;2320.\u003c/li\u003e\n\u003cli\u003ePonnanikajamideen M, et al. Sodium alginate\u0026ndash;Ag nanocomposite hydrogels for wound-healing and reusability. Carbohydr Polym. 2021;257:117603.\u003c/li\u003e\n\u003cli\u003eVarma RS. 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Structural and Morphological Analysis of Bio- Synthesized AgNPs and Their Antimicrobial Efficacy. J Environ Chem Eng. 2023;11(3):110945. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jece.2023.110945\u003c/span\u003e\u003c/span\u003e.\u003c/li\u003e\n\u003cli\u003eVarma RS. Green Nanochemistry: Design, Synthesis, and Applications of Nanoparticles with Controlled Release and Functional Stability. ACS Omega. 2023;8(6):5500\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsomega.4c11045\u003c/span\u003e\u003c/span\u003e.\u003c/li\u003e\n\u003cli\u003eZhao T, Huang Q, Liu D. \u003cem\u003ePolymer\u0026ndash;Metal Hybrid Nanocomposites: Mechanisms of\u0026nbsp;\u003c/em\u003eIon Release and Antimicrobial Action. Mater Adv, 2023, 4, 1912\u0026ndash;23.\u0026nbsp;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3MA00245G\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Premna serratifolia, green synthesis, Alginate hydrogel, silver nanocomposite, antimicrobial activity, Biopolymer, Sustained release, Eco-friendly nanotechnology","lastPublishedDoi":"10.21203/rs.3.rs-8189703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8189703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study reports the \u003cem\u003egreen fabrication\u003c/em\u003e of a sustainable \u003cb\u003ealginate\u0026ndash;silver nanocomposite hydrogel (Alg\u0026ndash;AgNC)\u003c/b\u003e employing the aqueous leaf extract of \u003cem\u003ePremna serratifolia\u003c/em\u003e L. as a natural reducing and stabilizing agent. The eco-friendly route enables in-situ formation of silver nanoparticles (AgNPs) within a sodium-alginate matrix, eliminating the need for chemical cross-linkers or toxic precursors. The developed nanocomposite was characterized using UV\u0026ndash;Vis spectroscopy, FTIR, XRD, SEM, TEM, and zeta-potential analysis. Comparative assessments between biogenic AgNPs and the Alg\u0026ndash;AgNC hydrogel evaluated improvements in physicochemical stability, structural integrity, and antimicrobial performance. The Alg\u0026ndash;AgNC hydrogel exhibited a surface-plasmon resonance at 428 nm, characteristic Ag (111) reflections in XRD, and uniformly dispersed nanoparticles (20\u0026ndash;40 nm) embedded within the alginate network. The zeta potential (\u0026ndash;32 mV) and FTIR spectra confirmed enhanced colloidal stability and effective phytochemical capping. Antibacterial testing against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e revealed a 1.5\u0026ndash;2-fold increase in inhibition-zone diameter compared with free AgNPs, attributed to sustained Ag⁺ release and synergistic polymer\u0026ndash;phytochemical interactions. The composite hydrogel remained stable for over 30 days without aggregation, demonstrating mechanical robustness and reusability. This study presents the \u003cb\u003efirst report of an alginate\u0026ndash;silver nanocomposite hydrogel synthesized using\u003c/b\u003e \u003cb\u003ePremna serratifolia\u003c/b\u003e \u003cb\u003eleaf extract\u003c/b\u003e, offering a superior, biocompatible, and sustainable nanomaterial platform for biomedical and environmental applications (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Green Fabrication of Alginate–Silver Nanocomposite Hydrogel Using Premna serratifolia Leaf Extract: Characterization, Comparative Evaluation, and Enhanced Antimicrobial Efficacy”","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 15:02:32","doi":"10.21203/rs.3.rs-8189703/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70d8a79a-6eea-4609-98bd-f61ebe43e2cc","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T15:56:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 15:02:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8189703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8189703","identity":"rs-8189703","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}