Biosynthesis of zinc oxide nanoparticles via neem extract and their anticancer and antibacterial activities

Abstract

In the present study, zinc oxide nanoparticles (ZnO-NPs) were synthesized using neem leaf aqueous extracts and characterized using transmission electron microscopy (TEM), ultraviolet visible spectroscopy (UV-Vis), and dynamic light scattering (DLS). Then compare its efficacy as anticancer and antibacterial agents with chemically synthesized ZnO-NPs and the neem leaf extract used for the green synthesis of ZnO-NPs. The TEM, UV-vis, and particle size confirmed that the developed ZnO-NPs are nanoscale. The chemically and greenly synthesized ZnO-NPs showed their optical absorbance at 328 nm and 380 nm, respectively, and were observed as spherical particles with a size of about 85 nm and 62.5 nm, respectively. HPLC and GC-MS were utilized to identify the bioactive components in the neem leaf aqueous extract employed for the eco-friendly production of ZnO-NPs. The HPLC analysis revealed that the aqueous extract of neem leaf contains 19 phenolic component fractions. The GC-MS analysis revealed the existence of 21 bioactive compounds. The antiproliferative effect of green ZnO-NPs was observed at different concentrations (31.25 µg/mL-1000 µg/mL) on Hct 116 and A 549 cancer cells, with an IC50 value of 111 µg/mL for A 549 and 118 µg/mL for Hct 116. On the other hand, the antibacterial activity against gram-positive and gram-negative bacteria was estimated. The antibacterial result showed that the MIC of green synthesized ZnO-NPs against gram-positive and gram-negative bacteria were 5, and 1 µg/mL. Hence, they could be utilized as effective antibacterial and antiproliferative agents.

Keywords: Antibacterial; Anticancer; Green nanoparticles; Neem; Phenolic compounds; ZnO-NPs.

Figure 1. Transmission electron microscope (A), absorbance of ZnO-NPs UV-VS (B) particle size (C), and zeta potential (D) of ZnO NPs prepared using chemical method.

Figure 2. Transmission electron microscope (A), absorbance of ZnO NPs UV-VIS (B) particle size (C), and zeta potential (D) of ZnO-NPs prepared using green synthesis.

Figure 3. GC-MS chromatogram of neem leaves ethyl acetate extract.

Figure 4. The shape of HCT 116 cells changed after being treated for 24 h with neem aqueous extract, Zn-NPs, and green synthesized ZnO-NPs at IC₅₀ concentrations (350, 190, and 118 µg/mL, respectively).

Figure 5. The shape of A549 cells changed after being treated for 24 h with neem aqueous extract, Zn-NPs, and green synthesized ZnO-NPs at IC₅₀ concentrations (227, 197, and 111 µg/mL, respectively).

Figure 6. Cell viability (%) and Toxicity (%) of A549 (A), and HCT 116 (B) cell lines treated with ZnO-NPs, neem aqueous extract, and green synthesized ZnO-NPs at different concentrations.

Different letters indicate significant differences among the cell viability (small letters) and toxicity (capital letters) according to Tukey’s HSD test (p ≤ 0.05).

Figure 7. The 50% inhibitory concentration (IC₅₀) values of ZnO-NPs, neem extract, and green synthesized ZnO-NPs for A549 and HCT 116 cell lines after 24 hours of treatment.

The different letters indicate significant differences among the IC50 against HCT 116 (small letters) and the IC50 against A549 (capital letters), according to Tukey’s HSD test (p ≤ 0.05).

Figure 8. Effect of neem aqueous extract, ZnO-NPs, and green synthesized ZnO-NPs on caspase 9 gene expression of two human cancer cell lines (A549, and HCT 116).

Cells were treated with the concentration causing the IC₅₀ for each cell line for 24 h and their mRNA levels were evaluated by quantitative real-time PCR. The different letters indicate significant differences among treatments, according to Tukey’s HSD test (p ≤ 0.05).

Figure 9. Transmission electron microscopy graphs Staph. aureus, and E. coli as subjected to 1 MIC of neem aqueous extract, ZnO-NPs, and green synthesized ZnO-NPs.

Figure 10. The plausible mechanism of formation of zinc oxide nanoparticles (ZnO-NPs) from neem aqueous leaf extract.