Green synthesis of copper oxide nanoparticles from Calotropis gigantea of antimicrobial, antidiabetic, and toxicity evaluation in zebrafish

Calotropis gigantea (giant milkweed or crown flower), a traditional medicinal plant used to treat various ailments, contains toxic compounds that require careful handling. In this study, copper oxide nanoparticles (CuO NPs) were synthesized using an eco-friendly green method with C. gigantea leaf ethanol extract ofsynthesized and characterized copper oxide nanoparticles (CG-CuO NPs) using UV–Vis spectrophotometry, FT-IR, XRD, SEM, and EDX. The study evaluated the in vitro bioactivity of the CG-CuO NPs leaf extract focusing on their antidiabetic, anti-inflammatory, cytotoxic, and antimicrobial properties. The Phytochemical screening of secondary metabolites such as flavonoids, tannins, steroids, saponins, polyphenols which act as efficient bio-reductants and capping agents, leading to smaller, more stable CuO nanoparticles with superior bioactivity. Characterization and confirmation of CG-CuO NPs synthesis by UV–Visible spectroscopy confirmed nanoparticle formation, showing a characteristic absorbance peak at 437 nm. Stabilization analysis of FTIR analysis identified hydroxyl, amine, carboxyl, and aromatic functional groups involved in nanoparticle stabilization, with prominent absorption bands at 1072 cm⁻¹ and 530 cm⁻¹. Morphology and size of SEM revealed predominantly spherical nanoparticles with smooth surfaces and uniform dispersion. The TEM images showed that the particle sizes ranged from 35 to 85 nm, following a narrow quasi-Gaussian size distribution. The crystallinity and Composition of XRD analysis confirmed the crystalline monoclinic phase of the CuO NPs (matching JCPDS No. 48–1548). Elemental analysis indicated that copper oxide was the major component (75.37 wt%), with trace amounts of carbon, sodium, and oxygen. Toxicity analysis of zebrafish embryotoxicity assays showed concentration- and time-dependent developmental toxicity, including deformities and mortality. Decreasing LC₅₀ and EC₅₀ values between 24- and 120-h post-fertilization indicated increasing toxicity over time. Biological Activities of the biogenic CuO NPs exhibited strong biological effects, including potent α-glucosidase inhibition (IC₅₀ = 22 μg/mL). The significant anti-inflammatory activity (IC₅₀ = 109 μg/mL) exceeded that of the chemically synthesized counterparts. The CG-CuO NPs exhibited selective anticancer potential, with an IC₅₀ of 31.5 μg/mL against MCF-7 breast cancer cells compared to 124.8 μg/mL in non-cancerous HaCaT keratinocytes, demonstrating preferential cytotoxicity toward cancer cells. C. gigantea-synthesized CuO nanoparticles demonstrate promising antidiabetic, anti-inflammatory, and selective anticancer properties of green synthesis and biocompatibility supporting potential biomedical applications.