# Green Synthesis of Copper Oxide Nanoparticles Using Citrus sinensis Leaves: Effects of Experimental Parameters, Antimicrobial Evaluation and Development of Chitosan Composites

**Authors:** Jordana Bortoluz, Axel J. P. Jacquot, Lucas C. Colissi, Paula Sartori, Lílian V. R. Beltrami, Régis Guégan, Giovanna Machado, Mariana Roesch-Ely, Janaina S. Crespo, Marcelo Giovanela

PMC · DOI: 10.3390/nano16060369 · Nanomaterials · 2026-03-18

## TL;DR

This paper describes a green method to synthesize copper oxide nanoparticles using orange leaves, which are then combined with chitosan to create antimicrobial composites.

## Contribution

A sustainable, green synthesis method for CuONPs using Citrus sinensis leaf extract and their integration into chitosan composites is proposed.

## Key findings

- CuONPs synthesized at pH 7.0, 10.0 g L−1 Cu(CH3COO)2·H2O, and 300 °C calcination showed antimicrobial activity against E. coli and S. aureus.
- Chitosan composites with CuONPs exhibited enhanced antimicrobial performance compared to individual components.
- The green synthesis method provides a sustainable alternative to conventional toxic chemical-based approaches.

## Abstract

Copper oxide nanoparticles (CuONPs) have received considerable attention because of their wide range of applications, particularly in the development of antimicrobial materials for medical, environmental, and industrial purposes. However, conventional synthesis routes often involve the use of toxic chemicals and environmentally harmful conditions. To overcome these limitations, green synthesis strategies have been developed as sustainable alternatives through the use of natural reducing and stabilizing agents. In this study, Citrus sinensis leaf extract, which exhibits high antioxidant capacity, was investigated for green synthesis of CuONPs, followed by their subsequent incorporation into a chitosan polymeric matrix. The optimal synthesis conditions were achieved at a pH of 7.0 using copper(II) acetate monohydrate (Cu(CH3COO)2·H2O) at a concentration of 10.0 g L−1 and a calcination temperature of 300 °C. The resulting CuONPs exhibited a heterogeneous morphology, with average particle sizes ranging from 20 to 30 nm, and demonstrated satisfactory antimicrobial activity against Escherichia coli and Staphylococcus aureus. The incorporation of these NPs into chitosan yielded composite materials with enhanced antimicrobial performance, highlighting the added value of polymer–NP hybrid systems. Although these composite materials were not evaluated under realistic operational conditions, the optimized green protocol provides a robust methodological basis for future studies targeting water disinfection and other environmentally relevant technologies.

## Linked entities

- **Chemicals:** copper(II) acetate monohydrate (PubChem CID 165397), chitosan (PubChem CID 129662530)
- **Species:** Citrus sinensis (taxon 2711)

## Full-text entities

- **Diseases:** toxicity (MESH:D064420), injury to (MESH:D014947), fungal (MESH:D009181)
- **Chemicals:** acetic acid (MESH:D019342), ascorbic acid (MESH:D001205), metal (MESH:D008670), 1,1-diphenyl-2-picrylhydrazyl (MESH:C004931), flavonoids (MESH:D005419), cellulose (MESH:D002482), platinum (MESH:D010984), Chitosan (MESH:D048271), tannins (MESH:D013634), glutaraldehyde (MESH:D005976), NaOH (MESH:D012972), Cu (MESH:D003300), Au (MESH:D006046), poly(acrylic acid) (MESH:C006903), sodium borohydride (MESH:C025364), lipids (MESH:D008055), agar (MESH:D000362), Cu2O (MESH:C000520), sugars (MESH:D000073893), essential oils (MESH:D009822), water (MESH:D014867), alumina (MESH:D000537), isopropanol (MESH:D019840), GA (MESH:D005707), polymer (MESH:D011108), ROS (MESH:D017382), Cu(I) (MESH:C073870), C. sinensis (-), CuO (MESH:C030973), hydrazine (MESH:C029424), saponins (MESH:D012503), copper(II) acetate monohydrate (MESH:C015092), molybdenum (MESH:D008982), poly(allylamine hydrochloride) (MESH:C063994), KCl (MESH:D011189), silicon (MESH:D012825), NP (MESH:D009405), sodium carbonate (MESH:C005686), terpenoids (MESH:D013729), Cu(OH)2 (MESH:C001606), ethanol (MESH:D000431), vancomycin (MESH:D014640), CuCl2 (MESH:C029892), O (MESH:D010100), phenols (MESH:D010636), C (MESH:D002244), methanol (MESH:D000432), salt (MESH:D012492), eriocitrin (MESH:C114706), anthocyanins (MESH:D000872), acetone (MESH:D000096)
- **Species:** Hyphaene thebaica (species) [taxon 115479], C. sinensis [taxon 128511], Citrus x clementina (clementine, species) [taxon 85681], Escherichia coli (E. coli, species) [taxon 562], Streptococcus agalactiae (species) [taxon 1311], Origanum majorana (sweet marjoram, species) [taxon 268884], Homo sapiens (human, species) [taxon 9606], Enterococcus (genus) [taxon 1350], Punica granatum (granado, species) [taxon 22663], Camellia sinensis (black tea, species) [taxon 4442], Simarouba glauca (aceituno, species) [taxon 43729], Citrus x aurantium (bitter orange, species) [taxon 43166], Staphylococcus aureus (species) [taxon 1280], Enterobacter cloacae (species) [taxon 550], Berberis lycium (species) [taxon 659594], Citrus x limon (lemon, species) [taxon 2708], Citrus x aurantiifolia (lime, species) [taxon 159033], Citrus sinensis (apfelsine, species) [taxon 2711], Citrus jambhiri (citronelle, species) [taxon 64884], Bacteria Latreille et al. 1825 (Bacteria stick insect, genus) [taxon 629395], Allium sativum (garlic, species) [taxon 4682]
- **Cell lines:** ATCC, 25923 — Homo sapiens (Human), Lung adenocarcinoma, Cancer cell line (CVCL_0023)

## Full text

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## Figures

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## References

73 references — full list in the complete paper: https://tomesphere.com/paper/PMC13029666/full.md

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Source: https://tomesphere.com/paper/PMC13029666