Synthesis and optimization of zinc oxide nanoparticles using Kalanchoe pinnata towards the evaluation of its anti-inflammatory activity

Abstract

The green synthesis of Zinc oxide nanoparticles (ZnO NPs) using Kalanchoe pinnata leaf extract has been illustrated in detail. Size plays a very significant role in maintaining the pharmacokinetic behavior of nanoparticle and thus, the size of ZnO NPs was optimized statistically using response surface methodology (RSM) with the Central composite design. Nanoparticles were characterized using UV–Visible spectrophotometer (UV–Vis), X-ray diffraction (XRD), Scanning electron microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDAX), Selected area electron diffraction (SAED), Transmission electron microscopy (TEM), Zeta analysis and Fourier transform infrared spectroscopy (FT-IR) which demonstrated the synthesis of hexagonal, spherical, pure and stable ZnO NPs with average crystallite size of 24 nm. The biocompatibility of ZnO NPs was established using 3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay on RAW 264.7 murine macrophage model. Anti-inflammatory potential of ZnO NPs was investigated on LPS induced murine macrophage RAW 264.7 cells. Gene expression of pro-inflammatory cytokines like Tumor necrosis factor (TNF-α), Interleukins (IL-1β, IL-6), and cyclooxygenase (COX-2) enzyme were checked using real-time polymerase chain reaction (RT-PCR). Enzyme-linked immunosorbent assay (ELISA) was performed to determine the protein level of cytokines upon ZnO treatment and LPS induction. The nitric oxide levels were evaluated with the help of Griess reagent and also the anti-oxidant activity was accessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay.