Millet-Mediated Green Synthesis of Zinc Oxide Nanoparticles: Dual Antidiabetic and Antioxidant Mechanisms toward Nano-Nutraceutical Applications

Millet-Mediated Green Synthesis of Zinc Oxide Nanoparticles: Dual Antidiabetic and Antioxidant Mechanisms toward Nano-Nutraceutical Applications | 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 Article Millet-Mediated Green Synthesis of Zinc Oxide Nanoparticles: Dual Antidiabetic and Antioxidant Mechanisms toward Nano-Nutraceutical Applications P Naveen, Dr Gopi Mamidi, Dr Indira Priyadarsini This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8330787/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 Diabetes mellitus remains one of the leading global metabolic disorders, primarily due to insufficient insulin secretion and cellular resistance to insulin action [ 1 ]. Zinc plays an integral role in insulin storage and secretion, while its deficiency contributes to β-cell dysfunction and oxidative stress [ 2 , 3 ]. The present study explores a sustainable, food-based strategy for zinc delivery through the green synthesis of zinc oxide nanoparticles (ZnO-NPs) using extracts of millets—Eleusine coracana (finger millet), Pennisetum glaucum (pearl millet), and Panicum sumatrense (little millet)—rich in phenolics and flavonoids [ 4 , 5 ].Zinc acetate precursors were reduced by millet phytochemicals under alkaline conditions to yield crystalline ZnO-NPs (20–80 nm) with surface-bound organic layers confirmed by UV–Vis, FTIR, XRD, TEM, and XPS analyses [ 6 – 8 ]. In-vitro assays revealed dose-dependent inhibition of α-amylase, α-glucosidase, and DPP-IV enzymes, as well as enhanced glucose-stimulated insulin secretion and glucose uptake in β-cell and adipocyte models [ 9 , 10 ]. The nanoparticles also exhibited significant antioxidant activity and negligible cytotoxicity, highlighting their safety and efficacy for potential nutraceutical applications. This study introduces a novel, millet-derived, biocompatible ZnO nanoformulation that unites agricultural sustainability with therapeutic efficacy for diabetes management, representing a promising step toward food-based nanomedicine [ 11 – 15 ]. Biological sciences/Biochemistry Biological sciences/Biotechnology Physical sciences/Nanoscience and technology Biological sciences/Plant sciences Zinc oxide nanoparticles Millets Green synthesis Antidiabetic Phytochemical reduction Insulin secretion Antioxidant activity In-vitro evaluation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Introduction Diabetes mellitus, particularly type 2 diabetes (T2D), is a chronic metabolic disorder characterized by elevated fasting and post-prandial glucose levels caused by impaired insulin secretion and insulin resistance [ 1 ]. Zinc plays a crucial role in β-cell physiology—its ions facilitate insulin crystallization within secretory granules through the ZnT8 transporter and modulate insulin storage, maturation, and release [ 2 , 3 ]. Deficiency in zinc homeostasis has been correlated with defective insulin signaling, oxidative stress, and chronic hyperglycemia [ 4 , 5 ]. Zinc oxide nanoparticles (ZnO-NPs) have emerged as an efficient vehicle for zinc delivery due to their high surface reactivity and controlled dissolution properties [ 6 ]. In-vitro studies report improved glucose tolerance, increased insulin levels, and reduced oxidative damage following ZnO-NP exposure in pancreatic cell models [ 7 , 8 ]. However, conventional chemical syntheses employ harsh reducing agents and surfactants that limit biomedical compatibility [ 9 ]. Green synthesis routes employing plant extracts are sustainable alternatives, where phytochemicals such as flavonoids, polyphenols, and organic acids act simultaneously as reducing and capping agents [ 10 , 11 ]. Millets—nutri-cereals including Eleusine coracana (finger millet), Pennisetum glaucum (pearl millet), and Panicum sumatrense (little millet)—are rich in phenolic compounds, dietary fiber, and essential minerals, notably zinc [ 12 , 13 ]. Their consumption has been linked to lower glycemic index, delayed starch digestion, and improved antioxidant defense [ 14 , 15 ]. Despite these advantages, the integration of millet phytochemicals into nanoparticle synthesis and evaluation for antidiabetic efficacy remains largely unexplored [ 16 ]. Therefore, this study hypothesizes that millet extracts can biosynthesize ZnO nanoparticles through phytochemical reduction, producing biocompatible nanostructures that simultaneously (i) enhance insulin secretion and signaling, (ii) inhibit carbohydrate-digesting enzymes, and (iii) attenuate oxidative stress. We pursue three aims: (1) optimize and characterize millet-mediated ZnO-NPs; (2) define in-vitro antidiabetic mechanisms and safety windows; and (3) assess the antioxidant and enzyme inhibitory activity using standard in-vitro assays. To the best of our knowledge, this is the first report describing the green synthesis of zinc oxide nanoparticles using millet extracts as both reducing and capping agents, followed by comprehensive in-vitro antidiabetic and antioxidant evaluation. The study uniquely integrates food-derived phytochemistry with trace-metal nanotechnology, establishing a dual-mechanism, sustainable approach for diabetes management. The work advances sustainable nano-nutraceutical design by tying a culturally relevant, food-based synthesis route to mechanistic and cellular validation [ 17 – 19 ]. 2. Materials and Methods 2.1 Materials and Reagents Millet species—finger millet ( Eleusine coracana ), pearl millet ( Pennisetum glaucum ), and little millet ( Panicum sumatrense )—were collected during the Kharif season (August 2025) from cultivated agricultural fields in Chandragiri Mandal, Tirupati District, Andhra Pradesh, India (13.63° N, 79.42° E). Chandragiri Mandal is a recognized agrarian region where millets are routinely cultivated as seasonal food crops [ 12 – 15 ]. Prior permission was obtained from the respective farmers and landowners before sample collection. The collection of plant materials complied with local agricultural practices and institutional guidelines. No protected, wild, or endangered plant species were involved, and no special permits or licenses were required for the collection of cultivated millet grains. The collected millet grains were botanically identified and authenticated by Dr. A. Indira Priyadarsini , Department of Botany, Government Degree College (Autonomous), Nagari, Andhra Pradesh, India. Voucher specimens were prepared and deposited in the SVU Herbarium with the following accession numbers: E. coracana (GDC/MIL/2025/01), P. glaucum (GDC/MIL/2025/02), and P. sumatrense (GDC/MIL/2025/03), following standard botanical authentication procedures [ 16 ]. The grains were thoroughly cleaned with distilled water, shade-dried at room temperature for 72 h, pulverized using a laboratory grinder, and sieved to obtain a uniform powder (≤ 250 µm) prior to extraction [ 22 ]. Zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O, ≥ 99% purity), sodium hydroxide (NaOH), Folin–Ciocalteu reagent, aluminium chloride, and all other analytical-grade chemicals were purchased from Merck India Pvt. Ltd., Mumbai, India , and used without further purification. Ultrapure water (18.2 MΩ·cm resistivity) obtained from a Milli-Q purification system was used throughout all experiments [ 20 ]. INS-1 (rat pancreatic β-cell line), 3T3-L1 (mouse pre-adipocyte cell line), and Caco-2 (human intestinal epithelial cell line) were procured from the National Centre for Cell Science (NCCS), Pune, India , and cultured according to ATCC/NCCS recommended protocols [ 21 ]. 2.2 Preparation of Millet Extracts Ten grams of millet flour were extracted with 100 mL of distilled water or 70% ethanol at 60°C for 1 h under constant stirring [ 22 ]. The extract was centrifuged (8,000 g, 10 min), filtered (0.45 µm), adjusted to pH 7, and stored at 4°C for ≤ 72 h. Total Phenolic Content (TPC) was determined by the Folin–Ciocalteu method [ 23 ], and Total Flavonoid Content (TFC) by the aluminium-chloride colorimetric assay [ 24 ]. 2.3 Green Synthesis of ZnO Nanoparticles Following optimized phytochemical reduction methods [ 25 , 26 ], 50 mL of 0.05 M zinc acetate (80°C) was mixed dropwise with 50 mL millet extract (1:1 v/v). pH was adjusted to 10.5 ± 0.3 with 1 M NaOH and stirred 90 min. The white precipitate was aged 12 h, centrifuged (10,000 g, 15 min), washed with water and 70% ethanol, dried (60°C), and calcined (300°C, 2 h) to obtain crystalline ZnO powder. The powder was dispersed (1 mg/mL) and dialyzed (12–14 kDa) to remove unreacted species. 2.4 Characterization UV–Vis Spectroscopy : 200–800 nm range using Shimadzu UV-2600; λmax ≈ 370 nm [ 27 ]. FTIR : PerkinElmer Spectrum 100; 4000–400 cm⁻¹; Zn–O band ≈ 450 cm⁻¹. XRD : Cu Kα (λ = 1.5406 Å); 2θ 20–80°; phase identified vs JCPDS 36-1451 [ 48 ]. SEM/TEM : morphology and size distribution; HR-TEM for lattice fringes [ 50 , 51 ]. DLS/Zeta Potential : Malvern Zetasizer Nano ZS at 25°C; dispersion stability [ 52 ]. XPS and ICP-OES : surface chemistry and Zn²⁺ release profiling in simulated fluids [ 28 ]. 2.5 In-Vitro Antidiabetic and Antioxidant Assays α-Amylase Inhibition : DNSA colorimetric method; IC₅₀ vs acarbose [ 29 ]. α-Glucosidase Inhibition : p-nitrophenyl-α-D-glucopyranoside substrate; abs 405 nm [ 30 ]. DPP-IV Inhibition : Fluorometric kit (Sigma-Aldrich). Glucose-Stimulated Insulin Secretion (GSIS) : INS-1 cells treated with ZnO-NPs (0.5–25 µg/mL); insulin quantified by ELISA [ 31 ]. Glucose Uptake : 2-NBDG fluorescent probe in 3T3-L1 adipocytes (± 100 nM insulin) [ 32 ]. Oxidative Stress : ROS assay (DCFDA); SOD, CAT, and GPx activities colorimetrically [ 33 ]. Cytotoxicity : MTT assay on INS-1 and Caco-2 cells (24/48 h) to determine NOAEL [ 34 ]. 2.6 Statistical Analysis All experiments were performed in triplicate (n = 3) and data are expressed as mean ± SD. Normality was verified by the Shapiro–Wilk test; statistical significance was analyzed using one-way ANOVA followed by Tukey’s post-hoc test (p < 0.05). Power analysis (α = 0.05, β = 0.2) confirmed adequate replication [ 38 , 39 ]. GraphPad Prism 10 was used for data visualization [ 40 ]. 3. Results and Discussion 3.1 Visual Observation and UV–Visible Spectroscopy Upon addition of millet extract to zinc acetate, an immediate color change from colorless to milky white indicated nanoparticle formation. UV–Vis spectra exhibited a characteristic surface plasmon resonance (SPR) peak at 372 ± 2 nm , confirming ZnO nanoparticle synthesis [ 41 ]. The absorbance intensity increased with reaction time (0–120 min), plateauing at 90 min, indicating complete reduction. The λmax values (370–380 nm) matched previous reports for plant-mediated ZnO nanoparticles [ 42 , 43 ]. Among the three extracts, pearl millet–ZnO NPs showed slightly higher absorbance, suggesting more efficient reduction due to greater phenolic content [ 44 ]. 3.2 Fourier Transform Infrared (FTIR) Analysis FTIR spectra revealed characteristic peaks at 3380 cm⁻¹ (O–H stretch) , 1630 cm⁻¹ (C = O stretch) , and 450 cm⁻¹ (Zn–O stretch) , confirming the formation of ZnO [ 45 ]. Reduced intensity of C–O–C and amide I bands in post-synthesis spectra implied involvement of hydroxyl, carbonyl, and amide groups in reduction and capping [ 46 ]. These bound organics enhance biocompatibility and stability [ 47 ]. 3.3 X-Ray Diffraction (XRD) Analysis XRD diffractograms exhibited sharp peaks at 2θ = 31.7°, 34.4°, 36.2°, 47.5°, 56.6°, 62.8°, and 67.9° , corresponding to the (100), (002), (101), (102), (110), (103), and (112) planes of hexagonal wurtzite ZnO (JCPDS 36-1451) [ 48 ]. Absence of impurity peaks indicated high purity. Average crystallite sizes (Scherrer equation) ranged 22–38 nm , consistent with TEM results. The smallest size (≈ 22 nm) was observed for pearl millet ZnO NPs , correlating with higher flavonoid content [ 49 ]. 3.4 Morphological and Particle Size Analysis (SEM/TEM) SEM micrographs showed quasi-spherical and hexagonal ZnO NPs with mild aggregation, while TEM confirmed well-dispersed nanoparticles (20–80 nm) with visible lattice fringes (d-spacing ≈ 0.26 nm; (002) plane) and clear SAED ring patterns, verifying crystalline nature [ 50 , 51 ]. EDS spectra displayed Zn and O peaks with minimal C signals from surface-bound organics, confirming purity. 3.5 Dynamic Light Scattering (DLS) and Zeta Potential Hydrodynamic diameters (80–110 nm) exceeded TEM sizes due to hydration and organic shells. Zeta potential values (− 24 to − 32 mV) indicated high colloidal stability [ 52 ]. Controlled Zn²⁺ release (< 20% over 24 h) in simulated intestinal fluid suggests safe bioavailability and potential for oral nutraceutical use [ 53 ]. 3.6 In-Vitro Antidiabetic Activity Millet-derived ZnO NPs demonstrated dose-dependent inhibition of α-amylase and α-glucosidase enzymes, with IC₅₀ values of 52.3 ± 1.8 µg/mL and 61.7 ± 2.1 µg/mL , respectively, comparable to acarbose (49.6 ± 1.5 µg/mL) [ 54 ]. DPP-IV inhibition reached 68% at 100 µg/mL , suggesting multi-enzyme modulation potential. In INS-1 β-cells , ZnO NPs enhanced glucose-stimulated insulin secretion (GSIS) by 1.7-fold at 10 µg/mL (p < 0.01) without cytotoxicity, while 3T3-L1 adipocytes showed 1.6-fold higher glucose uptake at 25 µg/mL compared with untreated control [ 55 ]. These outcomes indicate improved β-cell function and insulin sensitivity. 3.7 Antioxidant and Cytoprotective Activity ZnO NPs displayed strong free-radical scavenging capacity in DPPH assays (IC₅₀ ≈ 40 µg/mL) and reduced intracellular ROS by ~ 40% in INS-1 cells. Treated cells showed significantly elevated SOD, CAT, and GPx activities , confirming oxidative stress attenuation [ 55 ]. Cytotoxicity assays indicated > 90% cell viability up to 25 µg/mL and a NOAEL (No-Observed-Adverse-Effect Level) of 50 µg/mL [ 34 ]. 3.8 Proposed Mechanism of Action The phytochemicals in millet extracts (ferulic acid, catechins, flavones) act as dual reducing and capping agents , stabilizing ZnO NPs and enhancing biological response. On exposure to pancreatic and adipocyte models, Zn²⁺ ions released from ZnO NPs stimulate insulin secretion , while surface polyphenols inhibit carbohydrate-hydrolyzing enzymes and quench intracellular ROS . Thus, the synergistic triad of Zn²⁺ availability, enzyme inhibition, and antioxidant protection underlies their antidiabetic efficacy [ 56 ]. 3.9 Comparative Novelty and Scientific Significance Compared with other plant-mediated ZnO NPs (from Moringa oleifera , Aloe vera , Camellia sinensis ), millet-derived ZnO NPs showed: Smaller size (22–38 nm vs 40–60 nm) Higher stability (ζ ≈ −30 mV vs − 18 mV) Superior α-amylase inhibition and GSIS enhancement Food-based, non-toxic synthesis pathway This first-ever report of millet-mediated ZnO nanoparticles bridges agricultural sustainability and therapeutic nanomedicine, establishing a green nano-nutraceutical route for diabetes management [ 57 , 58 ]. 4. Conclusion and Future Perspectives Millet-mediated green synthesis of zinc oxide nanoparticles (ZnO-NPs) successfully demonstrates a sustainable, food-based route for producing stable, biocompatible nanostructures with dual antidiabetic and antioxidant action. The phytochemical constituents of finger, pearl, and little millets served as effective natural reducing and capping agents, yielding hexagonal wurtzite ZnO nanocrystals with uniform morphology and surface-bound bio-organic layers. The synthesized nanoparticles showed significant in-vitro inhibition of α-amylase, α-glucosidase, and DPP-IV enzymes , enhanced glucose-stimulated insulin secretion in pancreatic β-cells, and improved glucose uptake in adipocyte models. Concurrent antioxidant and cytoprotective effects were observed through reduced intracellular ROS levels and elevated antioxidant enzyme activity, confirming their biological compatibility. These findings highlight the potential of millet-derived ZnO NPs as eco-friendly nano-nutraceuticals for the prevention and management of diabetes. Future Perspectives Advanced mechanistic validation: Further in-vitro studies using β-cell transcriptomic and proteomic profiling are needed to confirm up-regulation of ZnT8 , INS1 , and antioxidant pathway genes, clarifying molecular mechanisms of ZnO-NP action. Bioavailability and pharmacokinetics: Quantitative evaluation of Zn²⁺ absorption, release kinetics, and cellular uptake via ICP-MS or fluorescence-tagged tracers will establish dose–response correlations. Nanoformulation enhancement: Embedding these nanoparticles into biopolymer or starch-based matrices could produce controlled-release nutraceutical capsules with improved gastrointestinal stability. Comparative cereal exploration: Extending the green synthesis approach to other zinc-rich cereals (foxtail, barnyard, and sorghum) may help correlate phytochemical diversity with nanoparticle performance. In-vivo and clinical translation: Following ethical clearance, in-vivo studies using diabetic animal models should validate long-term glycemic benefits, bioavailability, and safety. Circular bioeconomy approach: Millet husk and bran residues can be utilized as low-cost, sustainable precursors for large-scale green nanomaterial production, supporting a zero-waste, SDG-aligned industrial process. Declarations Acknowledgements: The authors gratefully acknowledge the Department of Chemistry, Government Degree College (Autonomous), Nagari, Andhra Pradesh, India , for providing laboratory facilities and instrumentation support during this study. Ethical Statement This study did not involve any animal or human subjects. All experiments were performed in vitro under institutional biosafety regulations. 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ZnT8-Mediated Mechanisms in Diabetes. Trends Endocrinol. Metab. 34 , 45–60 (2023). Iravani, S. et al. Comparative Green Syntheses of ZnO Nanoparticles. Green. Chem. 22 , 2647–2666 (2020). Kannan, R. et al. Biofunctional Plant-Based ZnO Nanoparticles for Therapeutics. Nano Biomed. Eng. 17 , 1–15 (2025). Additional Declarations No competing interests reported. Supplementary Files 6supplementarydata.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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8330787","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":563674119,"identity":"f063d308-871d-4489-a4f6-90193ea3e8a8","order_by":0,"name":"P Naveen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDADCXYGxgdAmoePeC3MDMwGIC1spGhhkwAxCGoxZz9j+ODjDgZ7yWbutMqvOXYybAzMDx/dwKPFsifH2HDmGYbE2cy8227LbksGOozN2DgHjxaDA2lp0rxtDAlyIC2S25iBWnjYpPFqOf8s/fffNgZ7kJZiyW31RGi5kXyMmbGNgRHkMMaP2w4To+XxYcneNonEmc28m6UZtx3nYWMm5JfziY0ffrbZ2Esc79348ee2ant+9uaHj/FpgQJwjDAw84BJwsoRgPEHKapHwSgYBaNgxAAAWso/ztBsae8AAAAASUVORK5CYII=","orcid":"","institution":"Government Degree College (Autonomous)","correspondingAuthor":true,"prefix":"","firstName":"P","middleName":"","lastName":"Naveen","suffix":""},{"id":563674120,"identity":"59768d52-da92-444d-9267-92246632f553","order_by":1,"name":"Dr Gopi Mamidi","email":"","orcid":"","institution":"Dr. V.S.K. 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1","display":"","copyAsset":false,"role":"figure","size":601898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of green synthesis and proposed antidiabetic mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMillet phytochemicals (polyphenols/flavonoids) reduce Zn²⁺ from zinc acetate to form \u003cstrong\u003ewurtzite ZnO nanoparticles\u003c/strong\u003e and remain as a surface “phytochemical corona.” Orally applied NPs (in a nutraceutical context) release \u003cstrong\u003ebioavailable Zn²⁺\u003c/strong\u003e, enhancing \u003cstrong\u003eβ-cell insulin synthesis/secretion (ZnT8-linked)\u003c/strong\u003e and \u003cstrong\u003einsulin signaling\u003c/strong\u003e in peripheral cells, while surface polyphenols \u003cstrong\u003einhibit α-amylase/α-glucosidase/DPP-IV\u003c/strong\u003e and \u003cstrong\u003equench intracellular ROS\u003c/strong\u003e, jointly improving glycemic control.\u003cstrong\u003e Panels:\u003c/strong\u003e (a) Green synthesis route; (b) nanoparticle with organic corona; (c) tri-pathway model: Zn²⁺ support + enzyme inhibition + antioxidant protection.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/971c63c2973f967e6c9217b8.png"},{"id":98796332,"identity":"4fca0b3a-ece5-451a-a82d-5b91839647b3","added_by":"auto","created_at":"2025-12-22 12:56:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV–Vis formation kinetics and FTIR evidence of phytochemical capping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cstrong\u003eUV–Vis time-course (0–120 min)\u003c/strong\u003eshowing growth of the excitonic band (λ_max ≈ 370–375 nm) until reaction plateau (~90 min). (b) Overlay of final spectra for \u003cstrong\u003efinger, pearl, little\u003c/strong\u003emillet syntheses. (c) \u003cstrong\u003eFTIR overlays\u003c/strong\u003e of extract vs ZnO-NPs: O–H (~3380 cm⁻¹), C=O (~1630 cm⁻¹) decrease/shift after synthesis; Zn–O band (~450 cm⁻¹) appears. (d) Band-assignment table supporting \u003cstrong\u003ephytochemical binding\u003c/strong\u003e on ZnO surfaces.\u003cbr\u003e\n \u003cstrong\u003eNotes:\u003c/strong\u003e mean of triplicate batches; shaded band = SD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/9d5ac1679e3c86d879aa5a92.png"},{"id":98796255,"identity":"c298f592-7daf-487e-bd44-0cd013b94e78","added_by":"auto","created_at":"2025-12-22 12:55:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":135707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD crystallinity and size; SEM/TEM morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cstrong\u003eXRD patterns (20–80° 2θ)\u003c/strong\u003e match hexagonal \u003cstrong\u003ewurtzite ZnO (JCPDS 36-1451)\u003c/strong\u003e with no impurity peaks. (b) \u003cstrong\u003eScherrer crystallite size\u003c/strong\u003e(22–38 nm) across millets. (c–e) \u003cstrong\u003eTEM images\u003c/strong\u003e (representative fields) reveal near-spherical/hexagonal ZnO (20–80 nm) with clear lattice fringes (d ≈ 0.26 nm, (002)); \u003cstrong\u003escale bars: 100 nm (TEM), 200 nm (SEM)\u003c/strong\u003e. (f) \u003cstrong\u003eSize histogram\u003c/strong\u003e from TEM (n ≥ 200 particles).\u003cstrong\u003e Stats:\u003c/strong\u003e mean ± SD; one-way ANOVA with Tukey, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3a.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/dbbb644a9f17e3c6ce34360e.png"},{"id":98796333,"identity":"2bbe93a0-5cfe-497c-bd5d-81f33dc44b2f","added_by":"auto","created_at":"2025-12-22 12:56:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScherrer Crystallite Size Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAverage Crystallite Size of ZnO Nanoparticles Synthesized Using Different Millet Extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBar graph representing the average crystallite size of ZnO nanoparticles calculated from XRD data using the Scherrer equation (D = Kλ/βcosθ). The values range from \u003cstrong\u003e22–38 nm\u003c/strong\u003e across finger, pearl, and little millet extracts, indicating the influence of phytochemical composition\u003c/p\u003e\n\u003cp\u003eon nucleation and growth.\u003cbr\u003e\n \u003cstrong\u003eStats:\u003c/strong\u003e mean ± SD, n = 3; ANOVA p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3b.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/8770d5f2340325429f60261b.png"},{"id":98797860,"identity":"b2e27cea-600a-4226-9107-e94e2db265fc","added_by":"auto","created_at":"2025-12-22 13:58:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":238022,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission Electron Micrograph (TEM) of ZnO Nanoparticles\u003c/p\u003e\n\u003cp\u003eTEM image displaying \u003cstrong\u003ewell-dispersed wurtzite ZnO nanoparticles\u003c/strong\u003e with average sizes ranging between \u003cstrong\u003e20–80 nm\u003c/strong\u003e. The particles exhibit \u003cstrong\u003equasi-spherical to hexagonal geometry\u003c/strong\u003e and distinct electron-dense boundaries corresponding to phytochemical stabilization.\u003cbr\u003e\n \u003cstrong\u003eScale bar:\u003c/strong\u003e 100 nm\u003c/p\u003e","description":"","filename":"3c.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/8ab627ff707a930cc1149aca.png"},{"id":98796329,"identity":"2fbc8b59-b1b3-4893-94ed-8b3104ddc1b6","added_by":"auto","created_at":"2025-12-22 12:56:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":392700,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Micrograph (SEM) of Millet-Mediated ZnO Nanoparticles\u003c/p\u003e\n\u003cp\u003eSEM image showing \u003cstrong\u003equasi-spherical and polygonal ZnO nanoparticles\u003c/strong\u003e with mild agglomeration and uniform distribution. The surface appears rough and textured, consistent with phytochemical capping and partial clustering typical of \u003cstrong\u003ebiogenic ZnO nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esynthesized via millet extracts.\u003cbr\u003e\n \u003cstrong\u003eScale bar:\u003c/strong\u003e 200 nm.\u003c/p\u003e","description":"","filename":"3d.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/37317c99944071f0ac137524.png"},{"id":98796313,"identity":"de8a9bf6-c78a-4240-91d2-e34026818652","added_by":"auto","created_at":"2025-12-22 12:56:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":304773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-Resolution TEM (HRTEM) Micrograph of ZnO Nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-resolution TEM image showing \u003cstrong\u003eclear lattice fringes\u003c/strong\u003e with an interplanar spacing of \u003cstrong\u003e~0.26 nm\u003c/strong\u003e, corresponding to the \u003cstrong\u003e(002)\u003c/strong\u003e plane of \u003cstrong\u003ehexagonal wurtzite ZnO\u003c/strong\u003e. The image confirms crystalline ordering and nanoscale periodicity within individual particles.\u003cbr\u003e\n \u003cstrong\u003eScale bar:\u003c/strong\u003e 10 nm.\u003c/p\u003e","description":"","filename":"3e.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/62730354218f8ae3413857da.png"},{"id":98796411,"identity":"b20d43e9-d6aa-4c54-8865-cb6a45163e3f","added_by":"auto","created_at":"2025-12-22 12:56:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":82369,"visible":true,"origin":"","legend":"","description":"","filename":"3f.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/95af99f8f141c48f80922750.png"},{"id":98796301,"identity":"9d2fcacc-9771-45bc-8fa9-844676f57304","added_by":"auto","created_at":"2025-12-22 12:55:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":95508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4. Colloidal properties and simulated-fluid ion release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cstrong\u003eDLS size distributions\u003c/strong\u003e (water) with Z-avg 80–110 nm due to hydration/corona; (b) \u003cstrong\u003ezeta potential\u003c/strong\u003e in water and SIF (pH 6.8), ζ ≈ −24 to −32 mV indicating good stability; (c) \u003cstrong\u003eZn²⁺ release profiles\u003c/strong\u003e in SGF (pH 1.2) vs SIF (pH 6.8), \u0026lt;20% over 24 h in SIF; (d) \u003cstrong\u003ephotographs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eshowing dispersion stability (no visible sediment at 24 h).\u003cbr\u003e\n \u003cstrong\u003eNotes:\u003c/strong\u003e n=3 independent batches; mean ± SD.\u003c/p\u003e","description":"","filename":"4a.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/d8132e2161782d1c6fc1e91a.png"},{"id":98796277,"identity":"fa08c1ee-2411-4627-ba3e-aef140338ff0","added_by":"auto","created_at":"2025-12-22 12:55:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":119889,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of millet extract and synthesized ZnO nanoparticles.\u003c/p\u003e\n\u003cp\u003eThe broad O–H stretching band (3380 cm⁻¹) and C=O (1630 cm⁻¹) decrease in intensity after synthesis, indicating involvement of hydroxyl and carbonyl groups in reduction and stabilization. A new band at ~450 cm⁻¹ corresponds to Zn–O stretching, confirming nanoparticle formation and phytochemical capping.\u003c/p\u003e","description":"","filename":"4b.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/dcc60be3055c1607420588a9.png"},{"id":98796666,"identity":"39fd61ca-b7fb-46ae-9fb8-508c5c824249","added_by":"auto","created_at":"2025-12-22 12:56:32","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":86457,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray Photoelectron Spectroscopy (XPS) spectrum of the Zn 2p region for millet-mediated ZnO nanoparticles. The prominent peak at \u003cstrong\u003e≈ 1021.5 eV\u003c/strong\u003ecorresponds to \u003cstrong\u003eZn 2p₃⁄₂\u003c/strong\u003e, confirming the presence of \u003cstrong\u003eZn²⁺\u003c/strong\u003echaracteristic of wurtzite ZnO. The absence of additional peaks indicates high purity and complete oxidation of zinc species.\u003c/p\u003e","description":"","filename":"4c.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/30e6b3292ada8fcb5fe79ab3.png"},{"id":98796264,"identity":"70441aa4-6bd7-42a2-8849-4ef41c9b5bf8","added_by":"auto","created_at":"2025-12-22 12:55:54","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":198774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-Resolution TEM (HRTEM) Micrograph of ZnO Nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-resolution TEM image showing \u003cstrong\u003eclear lattice fringes\u003c/strong\u003e with an interplanar spacing of \u003cstrong\u003e~0.26 nm\u003c/strong\u003e, corresponding to the \u003cstrong\u003e(002)\u003c/strong\u003e plane of \u003cstrong\u003ehexagonal wurtzite ZnO\u003c/strong\u003e. The image confirms crystalline ordering and nanoscale periodicity within individual particles.\u003cbr\u003e\n \u003cstrong\u003eScale bar:\u003c/strong\u003e 10 nm.\u003c/p\u003e","description":"","filename":"4d.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/3c4a9250176b3d26fb1ded4c.png"},{"id":98796675,"identity":"8db0d35e-c328-439d-8931-0701e7997e38","added_by":"auto","created_at":"2025-12-22 12:56:33","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":96734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5. In-vitro antidiabetic enzyme inhibition and cellular insulin biology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cstrong\u003eα-Amylase inhibition\u003c/strong\u003e: dose–response curves; inset IC₅₀ (µg/mL) vs acarbose control. (b) \u003cstrong\u003eα-Glucosidase inhibition\u003c/strong\u003e(pNPG assay) with IC₅₀ comparison. (c) \u003cstrong\u003eDPP-IV inhibition\u003c/strong\u003e (% at indicated doses). (d) \u003cstrong\u003eGSIS in INS-1 β-cells\u003c/strong\u003e: fold-change at 16.7 mM glucose ± ZnO-NPs (0.5–25 µg/mL), normalized to protein. (e) \u003cstrong\u003e2-NBDG uptake in 3T3-L1 adipocytes\u003c/strong\u003e: fold-increase ± 100 nM insulin.\u003cstrong\u003e Stats:\u003c/strong\u003e mean ± SD (n=3); one-way ANOVA/Tukey; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs\u003c/p\u003e\n\u003cp\u003econtrol; “ns” = not significant.\u003cstrong\u003e Cytotoxicity control:\u003c/strong\u003e parallel MTT confirms \u0026gt;90% viability ≤25 µg/mL.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/bf6234be7a7ecdf43d13bfd2.png"},{"id":98796154,"identity":"60adeb8a-84e5-4d65-855e-95398663988f","added_by":"auto","created_at":"2025-12-22 12:55:46","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":105852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 6. Antioxidant response and cytocompatibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cstrong\u003eIntracellular ROS (DCFDA)\u003c/strong\u003e in INS-1 cells: % of control decreases with ZnO-NP exposure (0–25 µg/mL). (b–d) \u003cstrong\u003eAntioxidant enzymes\u003c/strong\u003e (SOD, CAT, GPx) activities increased vs control. (e) \u003cstrong\u003eMTT viability\u003c/strong\u003e in INS-1 and Caco-2 cells (24/48 h): NOAEL defined at 50 µg/mL.\u003cbr\u003e\n\u003cstrong\u003eStats:\u003c/strong\u003e mean ± SD (n=3); ANOVA/Tukey; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/b4554c34abbfad9b81e8fb8e.png"},{"id":99791311,"identity":"bc655470-a046-4554-a58a-a8b4f9681a6d","added_by":"auto","created_at":"2026-01-08 12:59:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4826716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/2978bc09-965e-49e6-864a-cdd9c87d5350.pdf"},{"id":98797754,"identity":"332ff387-0256-4b50-8f62-ddc91cf57228","added_by":"auto","created_at":"2025-12-22 13:51:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9920866,"visible":true,"origin":"","legend":"","description":"","filename":"6supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/f529e7b70123458dc7ef75a7.docx"},{"id":98796341,"identity":"ef68762e-0e0b-46be-b279-6166dbec8b91","added_by":"auto","created_at":"2025-12-22 12:56:03","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":807366,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-8330787/v1/2bd149fb96f96f1423800e10.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Millet-Mediated Green Synthesis of Zinc Oxide Nanoparticles: Dual Antidiabetic and Antioxidant Mechanisms toward Nano-Nutraceutical Applications","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetes mellitus, particularly type 2 diabetes (T2D), is a chronic metabolic disorder characterized by elevated fasting and post-prandial glucose levels caused by impaired insulin secretion and insulin resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Zinc plays a crucial role in β-cell physiology\u0026mdash;its ions facilitate insulin crystallization within secretory granules through the ZnT8 transporter and modulate insulin storage, maturation, and release [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Deficiency in zinc homeostasis has been correlated with defective insulin signaling, oxidative stress, and chronic hyperglycemia [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Zinc oxide nanoparticles (ZnO-NPs) have emerged as an efficient vehicle for zinc delivery due to their high surface reactivity and controlled dissolution properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In-vitro studies report improved glucose tolerance, increased insulin levels, and reduced oxidative damage following ZnO-NP exposure in pancreatic cell models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, conventional chemical syntheses employ harsh reducing agents and surfactants that limit biomedical compatibility [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Green synthesis routes employing plant extracts are sustainable alternatives, where phytochemicals such as flavonoids, polyphenols, and organic acids act simultaneously as reducing and capping agents [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Millets\u0026mdash;nutri-cereals including \u003cem\u003eEleusine coracana\u003c/em\u003e (finger millet), \u003cem\u003ePennisetum glaucum\u003c/em\u003e (pearl millet), and \u003cem\u003ePanicum sumatrense\u003c/em\u003e (little millet)\u0026mdash;are rich in phenolic compounds, dietary fiber, and essential minerals, notably zinc [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Their consumption has been linked to lower glycemic index, delayed starch digestion, and improved antioxidant defense [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Despite these advantages, the integration of millet phytochemicals into nanoparticle synthesis and evaluation for antidiabetic efficacy remains largely unexplored [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, this study hypothesizes that millet extracts can biosynthesize ZnO nanoparticles through phytochemical reduction, producing biocompatible nanostructures that simultaneously (i) enhance insulin secretion and signaling, (ii) inhibit carbohydrate-digesting enzymes, and (iii) attenuate oxidative stress. We pursue three aims: (1) optimize and characterize millet-mediated ZnO-NPs; (2) define in-vitro antidiabetic mechanisms and safety windows; and (3) assess the antioxidant and enzyme inhibitory activity using standard in-vitro assays.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTo the best of our knowledge, this is the first report describing the green synthesis of zinc oxide nanoparticles using millet extracts as both reducing and capping agents, followed by comprehensive in-vitro antidiabetic and antioxidant evaluation. The study uniquely integrates food-derived phytochemistry with trace-metal nanotechnology, establishing a dual-mechanism, sustainable approach for diabetes management.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe work advances sustainable nano-nutraceutical design by tying a culturally relevant, food-based synthesis route to mechanistic and cellular validation [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e \u003cp\u003eMillet species\u0026mdash;finger millet (\u003cem\u003eEleusine coracana\u003c/em\u003e), pearl millet (\u003cem\u003ePennisetum glaucum\u003c/em\u003e), and little millet (\u003cem\u003ePanicum sumatrense\u003c/em\u003e)\u0026mdash;were collected during the Kharif season (August 2025) from cultivated agricultural fields in Chandragiri Mandal, Tirupati District, Andhra Pradesh, India (13.63\u0026deg; N, 79.42\u0026deg; E). Chandragiri Mandal is a recognized agrarian region where millets are routinely cultivated as seasonal food crops [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Prior permission was obtained from the respective farmers and landowners before sample collection. The collection of plant materials complied with local agricultural practices and institutional guidelines. No protected, wild, or endangered plant species were involved, and no special permits or licenses were required for the collection of cultivated millet grains.\u003c/p\u003e \u003cp\u003eThe collected millet grains were botanically identified and authenticated by \u003cb\u003eDr. A. Indira Priyadarsini\u003c/b\u003e, Department of Botany, Government Degree College (Autonomous), Nagari, Andhra Pradesh, India. Voucher specimens were prepared and deposited in the SVU Herbarium with the following accession numbers: \u003cem\u003eE. coracana\u003c/em\u003e (GDC/MIL/2025/01), \u003cem\u003eP. glaucum\u003c/em\u003e (GDC/MIL/2025/02), and \u003cem\u003eP. sumatrense\u003c/em\u003e (GDC/MIL/2025/03), following standard botanical authentication procedures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe grains were thoroughly cleaned with distilled water, shade-dried at room temperature for 72 h, pulverized using a laboratory grinder, and sieved to obtain a uniform powder (\u0026le;\u0026thinsp;250 \u0026micro;m) prior to extraction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eZinc acetate dihydrate (Zn(CH₃COO)₂\u0026middot;2H₂O, \u0026ge; 99% purity), sodium hydroxide (NaOH), Folin\u0026ndash;Ciocalteu reagent, aluminium chloride, and all other analytical-grade chemicals were purchased from \u003cb\u003eMerck India Pvt. Ltd., Mumbai, India\u003c/b\u003e, and used without further purification. Ultrapure water (18.2 MΩ\u0026middot;cm resistivity) obtained from a Milli-Q purification system was used throughout all experiments [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eINS-1 (rat pancreatic β-cell line), 3T3-L1 (mouse pre-adipocyte cell line), and Caco-2 (human intestinal epithelial cell line) were procured from the \u003cb\u003eNational Centre for Cell Science (NCCS), Pune, India\u003c/b\u003e, and cultured according to ATCC/NCCS recommended protocols [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of Millet Extracts\u003c/h2\u003e \u003cp\u003eTen grams of millet flour were extracted with 100 mL of distilled water or 70% ethanol at 60\u0026deg;C for 1 h under constant stirring [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The extract was centrifuged (8,000 g, 10 min), filtered (0.45 \u0026micro;m), adjusted to pH 7, and stored at 4\u0026deg;C for \u0026le;\u0026thinsp;72 h.\u003c/p\u003e \u003cp\u003eTotal Phenolic Content (TPC) was determined by the \u003cb\u003eFolin\u0026ndash;Ciocalteu method\u003c/b\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and Total Flavonoid Content (TFC) by the \u003cb\u003ealuminium-chloride colorimetric assay\u003c/b\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Green Synthesis of ZnO Nanoparticles\u003c/h2\u003e \u003cp\u003eFollowing optimized phytochemical reduction methods [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 50 mL of 0.05 M zinc acetate (80\u0026deg;C) was mixed dropwise with 50 mL millet extract (1:1 v/v). pH was adjusted to 10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 with 1 M NaOH and stirred 90 min. The white precipitate was aged 12 h, centrifuged (10,000 g, 15 min), washed with water and 70% ethanol, dried (60\u0026deg;C), and calcined (300\u0026deg;C, 2 h) to obtain crystalline ZnO powder. The powder was dispersed (1 mg/mL) and dialyzed (12\u0026ndash;14 kDa) to remove unreacted species.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4 Characterization\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eUV\u0026ndash;Vis Spectroscopy\u003c/b\u003e: 200\u0026ndash;800 nm range using Shimadzu UV-2600; λmax\u0026thinsp;\u0026asymp;\u0026thinsp;370 nm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eFTIR\u003c/b\u003e: PerkinElmer Spectrum 100; 4000\u0026ndash;400 cm⁻\u0026sup1;; Zn\u0026ndash;O band\u0026thinsp;\u0026asymp;\u0026thinsp;450 cm⁻\u0026sup1;.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eXRD\u003c/b\u003e: Cu Kα (λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;); 2θ 20\u0026ndash;80\u0026deg;; phase identified vs JCPDS 36-1451 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSEM/TEM\u003c/b\u003e: morphology and size distribution; HR-TEM for lattice fringes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eDLS/Zeta Potential\u003c/b\u003e: Malvern Zetasizer Nano ZS at 25\u0026deg;C; dispersion stability [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eXPS and ICP-OES\u003c/b\u003e: surface chemistry and Zn\u0026sup2;⁺ release profiling in simulated fluids [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 In-Vitro Antidiabetic and Antioxidant Assays\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eα-Amylase Inhibition\u003c/b\u003e: DNSA colorimetric method; IC₅₀ vs acarbose [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eα-Glucosidase Inhibition\u003c/b\u003e: \u003cem\u003ep-nitrophenyl-α-D-glucopyranoside\u003c/em\u003e substrate; abs 405 nm [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eDPP-IV Inhibition\u003c/b\u003e: Fluorometric kit (Sigma-Aldrich).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGlucose-Stimulated Insulin Secretion (GSIS)\u003c/b\u003e: INS-1 cells treated with ZnO-NPs (0.5\u0026ndash;25 \u0026micro;g/mL); insulin quantified by ELISA [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGlucose Uptake\u003c/b\u003e: 2-NBDG fluorescent probe in 3T3-L1 adipocytes (\u0026plusmn;\u0026thinsp;100 nM insulin) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eOxidative Stress\u003c/b\u003e: ROS assay (DCFDA); SOD, CAT, and GPx activities colorimetrically [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eCytotoxicity\u003c/b\u003e: MTT assay on INS-1 and Caco-2 cells (24/48 h) to determine NOAEL [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll experiments were performed in triplicate (n\u0026thinsp;=\u0026thinsp;3) and data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Normality was verified by the Shapiro\u0026ndash;Wilk test; statistical significance was analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Power analysis (α\u0026thinsp;=\u0026thinsp;0.05, β\u0026thinsp;=\u0026thinsp;0.2) confirmed adequate replication [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. GraphPad Prism 10 was used for data visualization [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Visual Observation and UV\u0026ndash;Visible Spectroscopy\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUpon addition of millet extract to zinc acetate, an immediate color change from colorless to milky white indicated nanoparticle formation. UV\u0026ndash;Vis spectra exhibited a characteristic \u003cb\u003esurface plasmon resonance (SPR) peak at 372\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm\u003c/b\u003e, confirming ZnO nanoparticle synthesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The absorbance intensity increased with reaction time (0\u0026ndash;120 min), plateauing at 90 min, indicating complete reduction. The λmax values (370\u0026ndash;380 nm) matched previous reports for plant-mediated ZnO nanoparticles [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the three extracts, \u003cb\u003epearl millet\u0026ndash;ZnO NPs\u003c/b\u003e showed slightly higher absorbance, suggesting more efficient reduction due to greater phenolic content [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Fourier Transform Infrared (FTIR) Analysis\u003c/h2\u003e \u003cp\u003eFTIR spectra revealed characteristic peaks at \u003cb\u003e3380 cm⁻\u0026sup1; (O\u0026ndash;H stretch)\u003c/b\u003e, \u003cb\u003e1630 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O stretch)\u003c/b\u003e, and \u003cb\u003e450 cm⁻\u0026sup1; (Zn\u0026ndash;O stretch)\u003c/b\u003e, confirming the formation of ZnO [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Reduced intensity of C\u0026ndash;O\u0026ndash;C and amide I bands in post-synthesis spectra implied involvement of hydroxyl, carbonyl, and amide groups in reduction and capping [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These bound organics enhance biocompatibility and stability [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 X-Ray Diffraction (XRD) Analysis\u003c/h2\u003e \u003cp\u003eXRD diffractograms exhibited sharp peaks at \u003cb\u003e2θ\u0026thinsp;=\u0026thinsp;31.7\u0026deg;, 34.4\u0026deg;, 36.2\u0026deg;, 47.5\u0026deg;, 56.6\u0026deg;, 62.8\u0026deg;, and 67.9\u0026deg;\u003c/b\u003e, corresponding to the (100), (002), (101), (102), (110), (103), and (112) planes of \u003cb\u003ehexagonal wurtzite ZnO (JCPDS 36-1451)\u003c/b\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Absence of impurity peaks indicated high purity. Average crystallite sizes (Scherrer equation) ranged \u003cb\u003e22\u0026ndash;38 nm\u003c/b\u003e, consistent with TEM results. The smallest size (\u0026asymp;\u0026thinsp;22 nm) was observed for \u003cb\u003epearl millet ZnO NPs\u003c/b\u003e, correlating with higher flavonoid content [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Morphological and Particle Size Analysis (SEM/TEM)\u003c/h2\u003e \u003cp\u003eSEM micrographs showed \u003cb\u003equasi-spherical and hexagonal ZnO NPs\u003c/b\u003e with mild aggregation, while TEM confirmed \u003cb\u003ewell-dispersed nanoparticles (20\u0026ndash;80 nm)\u003c/b\u003e with visible lattice fringes (d-spacing\u0026thinsp;\u0026asymp;\u0026thinsp;0.26 nm; (002) plane) and clear SAED ring patterns, verifying crystalline nature [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. EDS spectra displayed Zn and O peaks with minimal C signals from surface-bound organics, confirming purity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Dynamic Light Scattering (DLS) and Zeta Potential\u003c/h2\u003e \u003cp\u003eHydrodynamic diameters (80\u0026ndash;110 nm) exceeded TEM sizes due to hydration and organic shells. Zeta potential values (\u0026minus;\u0026thinsp;24 to \u0026minus;\u0026thinsp;32 mV) indicated high colloidal stability [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Controlled Zn\u0026sup2;⁺ release (\u0026lt;\u0026thinsp;20% over 24 h) in simulated intestinal fluid suggests safe bioavailability and potential for oral nutraceutical use [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6 In-Vitro Antidiabetic Activity\u003c/h2\u003e \u003cp\u003eMillet-derived ZnO NPs demonstrated \u003cb\u003edose-dependent inhibition of α-amylase and α-glucosidase\u003c/b\u003e enzymes, with IC₅₀ values of \u003cb\u003e52.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 \u0026micro;g/mL\u003c/b\u003e and \u003cb\u003e61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u0026micro;g/mL\u003c/b\u003e, respectively, comparable to acarbose (49.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u0026micro;g/mL) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDPP-IV inhibition reached \u003cb\u003e68% at 100 \u0026micro;g/mL\u003c/b\u003e, suggesting multi-enzyme modulation potential. In \u003cb\u003eINS-1 β-cells\u003c/b\u003e, ZnO NPs enhanced \u003cb\u003eglucose-stimulated insulin secretion (GSIS)\u003c/b\u003e by \u003cb\u003e1.7-fold\u003c/b\u003e at 10 \u0026micro;g/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) without cytotoxicity, while \u003cb\u003e3T3-L1 adipocytes\u003c/b\u003e showed \u003cb\u003e1.6-fold higher glucose uptake\u003c/b\u003e at 25 \u0026micro;g/mL compared with untreated control [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These outcomes indicate improved β-cell function and insulin sensitivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Antioxidant and Cytoprotective Activity\u003c/h2\u003e \u003cp\u003eZnO NPs displayed \u003cb\u003estrong free-radical scavenging capacity\u003c/b\u003e in DPPH assays (IC₅₀ \u0026asymp; 40 \u0026micro;g/mL) and reduced intracellular ROS by ~\u0026thinsp;40% in INS-1 cells. Treated cells showed significantly elevated \u003cb\u003eSOD, CAT, and GPx activities\u003c/b\u003e, confirming oxidative stress attenuation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Cytotoxicity assays indicated\u0026thinsp;\u0026gt;\u0026thinsp;90% cell viability up to 25 \u0026micro;g/mL and a \u003cb\u003eNOAEL (No-Observed-Adverse-Effect Level)\u003c/b\u003e of 50 \u0026micro;g/mL [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Proposed Mechanism of Action\u003c/h2\u003e \u003cp\u003eThe phytochemicals in millet extracts (ferulic acid, catechins, flavones) act as \u003cb\u003edual reducing and capping agents\u003c/b\u003e, stabilizing ZnO NPs and enhancing biological response. On exposure to pancreatic and adipocyte models, Zn\u0026sup2;⁺ ions released from ZnO NPs \u003cb\u003estimulate insulin secretion\u003c/b\u003e, while surface polyphenols \u003cb\u003einhibit carbohydrate-hydrolyzing enzymes\u003c/b\u003e and \u003cb\u003equench intracellular ROS\u003c/b\u003e. Thus, the \u003cb\u003esynergistic triad of Zn\u0026sup2;⁺ availability, enzyme inhibition, and antioxidant protection\u003c/b\u003e underlies their antidiabetic efficacy [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Comparative Novelty and Scientific Significance\u003c/h2\u003e \u003cp\u003eCompared with other plant-mediated ZnO NPs (from \u003cem\u003eMoringa oleifera\u003c/em\u003e, \u003cem\u003eAloe vera\u003c/em\u003e, \u003cem\u003eCamellia sinensis\u003c/em\u003e), millet-derived ZnO NPs showed:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eSmaller size (22\u0026ndash;38 nm vs 40\u0026ndash;60 nm)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eHigher stability (ζ \u0026asymp; \u0026minus;30 mV vs \u0026minus;\u0026thinsp;18 mV)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSuperior α-amylase inhibition and GSIS enhancement\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFood-based, non-toxic synthesis pathway\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThis first-ever report of millet-mediated ZnO nanoparticles bridges agricultural sustainability and therapeutic nanomedicine, establishing a green nano-nutraceutical route for diabetes management [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion and Future Perspectives","content":"\u003cp\u003eMillet-mediated green synthesis of zinc oxide nanoparticles (ZnO-NPs) successfully demonstrates a sustainable, food-based route for producing stable, biocompatible nanostructures with dual \u003cb\u003eantidiabetic and antioxidant\u003c/b\u003e action. The phytochemical constituents of finger, pearl, and little millets served as effective natural reducing and capping agents, yielding hexagonal wurtzite ZnO nanocrystals with uniform morphology and surface-bound bio-organic layers.\u003c/p\u003e \u003cp\u003eThe synthesized nanoparticles showed \u003cb\u003esignificant in-vitro inhibition of α-amylase, α-glucosidase, and DPP-IV enzymes\u003c/b\u003e, enhanced \u003cb\u003eglucose-stimulated insulin secretion\u003c/b\u003e in pancreatic β-cells, and improved \u003cb\u003eglucose uptake\u003c/b\u003e in adipocyte models. Concurrent antioxidant and cytoprotective effects were observed through reduced intracellular ROS levels and elevated antioxidant enzyme activity, confirming their biological compatibility. These findings highlight the potential of millet-derived ZnO NPs as \u003cb\u003eeco-friendly nano-nutraceuticals\u003c/b\u003e for the prevention and management of diabetes.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFuture Perspectives\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eAdvanced mechanistic validation:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eFurther in-vitro studies using \u0026beta;-cell transcriptomic and proteomic profiling are needed to confirm up-regulation of \u003cem\u003eZnT8\u003c/em\u003e, \u003cem\u003eINS1\u003c/em\u003e, and antioxidant pathway genes, clarifying molecular mechanisms of ZnO-NP action.\u003c/p\u003e\n\u003col start=\"2\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eBioavailability and pharmacokinetics:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eQuantitative evaluation of Zn\u0026sup2;⁺ absorption, release kinetics, and cellular uptake via ICP-MS or fluorescence-tagged tracers will establish dose\u0026ndash;response correlations.\u003c/p\u003e\n\u003col start=\"3\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eNanoformulation enhancement:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eEmbedding these nanoparticles into biopolymer or starch-based matrices could produce \u003cstrong\u003econtrolled-release nutraceutical capsules\u003c/strong\u003e with improved gastrointestinal stability.\u003c/p\u003e\n\u003col start=\"4\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eComparative cereal exploration:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eExtending the green synthesis approach to other zinc-rich cereals (foxtail, barnyard, and sorghum) may help correlate phytochemical diversity with nanoparticle performance.\u003c/p\u003e\n\u003col start=\"5\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eIn-vivo and clinical translation:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eFollowing ethical clearance, in-vivo studies using diabetic animal models should validate long-term glycemic benefits, bioavailability, and safety.\u003c/p\u003e\n\u003col start=\"6\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eCircular bioeconomy approach:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eMillet husk and bran residues can be utilized as low-cost, sustainable precursors for large-scale green nanomaterial production, supporting a \u003cstrong\u003ezero-waste, SDG-aligned\u003c/strong\u003e industrial process.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the \u003cstrong\u003eDepartment of Chemistry, Government Degree College (Autonomous), Nagari, Andhra Pradesh, India\u003c/strong\u003e, for providing laboratory facilities and instrumentation support during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did \u003cstrong\u003enot\u003c/strong\u003e involve any animal or human subjects. All experiments were performed \u003cstrong\u003ein vitro\u003c/strong\u003e under institutional biosafety regulations. Future \u003cem\u003ein vivo\u003c/em\u003e validation will be conducted after obtaining ethical clearance from the Institutional Animal Ethics Committee (IAEC), Sri Venkateswara University, Tirupati, in compliance with CPCSEA guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received \u003cstrong\u003eno external funding\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare \u003cstrong\u003eno competing financial or non-financial interests\u003c/strong\u003e that could have influenced the results of this study.\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: Writing \u0026ndash; Original Draft:P.Naveen\u0026nbsp;\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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCho, N. H. et al. \u003cem\u003eIDF Diabetes Atlas\u003c/em\u003e 10th edn (International Diabetes Federation: Brussels,, 2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChimienti, F., Devergnas, S., Favier, A., Seve, M. \u0026amp; Zinc Pancreatic Islet Cell Function and Diabetes: New Insights into an Old Story. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A. 101\u003c/em\u003e, 9250\u0026ndash;9255. (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang, J., Li, M., Yang, Y., Xiao, W. \u0026amp; Shi, X. Zinc Transporter 8 (ZnT8): A Potential Target for Diabetes Therapy. \u003cem\u003eFront. 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Zeta Potential and Stability of Green ZnO Nanoparticles. \u003cem\u003eColloid Interface Sci. Commun.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e, 100618 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, M. et al. Controlled Zn\u0026sup2;⁺ Release from ZnO NPs in Gastrointestinal Media. \u003cem\u003eJ. Biomed. Nanotechnol\u003c/em\u003e. \u003cb\u003e20\u003c/b\u003e, 421\u0026ndash;430 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J. et al. α-Amylase and α-Glucosidase Inhibition by ZnO NPs. \u003cem\u003eBioNanoScience\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 889\u0026ndash;899 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik, R. et al. Antioxidant and GSIS Enhancement by ZnO Nanoparticles. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 897421 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanesan, K. et al. ZnT8-Mediated Mechanisms in Diabetes. \u003cem\u003eTrends Endocrinol. Metab.\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e, 45\u0026ndash;60 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIravani, S. et al. Comparative Green Syntheses of ZnO Nanoparticles. \u003cem\u003eGreen. Chem.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 2647\u0026ndash;2666 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKannan, R. et al. Biofunctional Plant-Based ZnO Nanoparticles for Therapeutics. \u003cem\u003eNano Biomed. Eng.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 1\u0026ndash;15 (2025).\u003c/span\u003e\u003c/li\u003e\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":"Zinc oxide nanoparticles, Millets, Green synthesis, Antidiabetic, Phytochemical reduction, Insulin secretion, Antioxidant activity, In-vitro evaluation","lastPublishedDoi":"10.21203/rs.3.rs-8330787/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8330787/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetes mellitus remains one of the leading global metabolic disorders, primarily due to insufficient insulin secretion and cellular resistance to insulin action [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Zinc plays an integral role in insulin storage and secretion, while its deficiency contributes to β-cell dysfunction and oxidative stress [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The present study explores a sustainable, food-based strategy for zinc delivery through the green synthesis of zinc oxide nanoparticles (ZnO-NPs) using extracts of millets\u0026mdash;Eleusine coracana (finger millet), Pennisetum glaucum (pearl millet), and Panicum sumatrense (little millet)\u0026mdash;rich in phenolics and flavonoids [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].Zinc acetate precursors were reduced by millet phytochemicals under alkaline conditions to yield crystalline ZnO-NPs (20\u0026ndash;80 nm) with surface-bound organic layers confirmed by UV\u0026ndash;Vis, FTIR, XRD, TEM, and XPS analyses [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In-vitro assays revealed dose-dependent inhibition of α-amylase, α-glucosidase, and DPP-IV enzymes, as well as enhanced glucose-stimulated insulin secretion and glucose uptake in β-cell and adipocyte models [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The nanoparticles also exhibited significant antioxidant activity and negligible cytotoxicity, highlighting their safety and efficacy for potential nutraceutical applications. This study introduces a novel, millet-derived, biocompatible ZnO nanoformulation that unites agricultural sustainability with therapeutic efficacy for diabetes management, representing a promising step toward food-based nanomedicine [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Millet-Mediated Green Synthesis of Zinc Oxide Nanoparticles: Dual Antidiabetic and Antioxidant Mechanisms toward Nano-Nutraceutical Applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 12:48:35","doi":"10.21203/rs.3.rs-8330787/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 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60022678,"name":"Biological sciences/Biochemistry"},{"id":60022679,"name":"Biological sciences/Biotechnology"},{"id":60022680,"name":"Physical sciences/Nanoscience and technology"},{"id":60022681,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-01-05T12:24:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 12:48:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8330787","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8330787","identity":"rs-8330787","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}