METHODS: Eucalyptol, a monoterpene oxide active, was used to formulate the NLC-Eu by using high pressure homogenization technique. The physicochemical characterization of NLC-Eu was performed to assess its morphology, particle size, polydispersity index, and zeta potential. The in vitro cytotoxic effects of this encapsulated eucalyptol on human (MDA MB-231) and murine (4 T1) breast cancer cell lines were determined using the MTT assay. Additionally, acridine orange/propidium iodide assay was conducted on the NLC-Eu treated MDA MB-231 cells. The in vivo sub-chronic toxicity of the prepared NLC-Eu was investigated using an in vivo BALB/c mice model.

RESULTS: As a result, the light, translucent, milky-colored NLC-Eu showed particle size of 71.800 ± 2.144 nm, poly-dispersity index of 0.258 ± 0.003, and zeta potential of - 2.927 ± 0.163 mV. Furthermore, the TEM results of NLC-Eu displayed irregular round to spherical morphology with narrow size distribution and relatively uniformed particles. The drug loading capacity and entrapment efficiency of NLC-Eu were 4.99 and 90.93%, respectively. Furthermore, NLC-Eu exhibited cytotoxic effects on both, human and mice, breast cancer cells with IC50 values of 10.00 ± 4.81 μg/mL and 17.70 ± 0.57 μg/mL, respectively at 72 h. NLC-Eu also induced apoptosis on the MDA MB-231 cells. In the sub-chronic toxicity study, all of the studied mice did not show any signs of toxicity, abnormality or mortality. Besides that, no significant changes were observed in the body weight, internal organ index, hepatic and renal histopathology, serum biochemistry, nitric oxide and malondialdehyde contents.

RESULTS: Having confirmed via histology, haematology and clinical biochemistry analyses that OPP is not toxic to mice, we further explored the gene expression changes caused by OPP through statistical and functional analyses using Illumina microarrays. OPP showed numerous biological activities in three major organs of mice, the liver, spleen and heart. In livers of mice given OPP, four lipid catabolism genes were up-regulated while five cholesterol biosynthesis genes were down-regulated, suggesting that OPP may play a role in reducing cardiovascular disease. OPP also up-regulated eighteen blood coagulation genes in spleens of mice. OPP elicited gene expression changes similar to the effects of caloric restriction in the hearts of mice supplemented with OPP. Microarray gene expression fold changes for six target genes in the three major organs tested were validated with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the correlation of fold changes obtained with these two techniques was high (R2 = 0.9653).

Methods: Twenty-four adult male Balb/C mice were divided into six groups: i) normal diet; ii) positive control diet; iii) negative control diet; and iv) HFD with SCGO at 12.5 mg/kg body weight (mg/kg BW); v) HFD with SCGO at 25 mg/kg BW, vi) HFD with SCGO at 50 mg/kg BW. Liver weight and morphology, spleen weight, serum levels of superoxide dismutase (SOD) and tumour necrosis factor α (TNF-α), TNF-α expression in the aorta and lipid profiles were assessed at the end of the experimental period.

Results: SCGO treatment was associated with significant decreases in liver and spleen weight as well as amelioration of hepatic steatosis. SCGO treatment also decreased TNF-α levels and expression. Serum levels of SOD in the SCGO groups were significantly increased compared with the negative control group. Lipid profiles were improved in the SCGO treatment groups compared with the negative control group.