Objective: We studied the cytotoxic effects of topical anise oil on NIH/3T3 fibroblast cells using a cell culture assay.
Materials and methods: NIH/3T3 fibroblast cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%) and penicillin/streptomycin under standard cell culture conditions in a humidified incubator containing 5% carbon dioxide. For the MTT cytotoxicity experiment, NIH/3T3 cells were plated in triplicate at a concentration of 3x103 per well in 96-well plates and incubated for 24 hours. The cells were treated with anise oil concentrations ranging from 3.13 to 100 μM, and the plates were cultured for 24, 48, and 72 hours under standard cell culture conditions. For assessment by confocal microscopy, NIH/3T3 cells were seeded on sterilized coverslips in 6-well plates at a concentration of 105 cells per well in triplicate. For 24 hours, cells were treated with 100 μM of anise oil. Three wells that were not treated with anise oil served as the control group.
Results: The MTT findings demonstrated that anise oil is not cytotoxic to NIH/3T3 fibroblast cells. Anise oil stimulated cell growth and triggered cell division at all three incubation intervals of 24, 48, and 72 hours. The maximum growth was obtained in the applied highest concentration of 100 μM anise oil. At doses of 25, 50, and 100 μM, there was also a statistically significant improvement in cell viability. At 72 hours of incubation, dosages of 6.25 and 12.5 micro of anise oil were shown to be viability-inducing for NIH/3T3 cells. In the confocal microscopy pictures, it was found that anise oil was not cytotoxic on NIH/3T3 cells at the applied maximal dose. The experimental group of NIH/3T3 cells exhibited the same cell morphology as the untreated control group. In both sets of NIH/3T3 cells, the nucleus was round and undamaged, and the cytoskeleton was determined to be compact.
Conclusions: Anise oil is not cytotoxic on NIH/3T3 fibroblast cells and initiates cell growth. Anise oil could be used topically to enhance wound healing after surgical procedures if clinical trials will confirm experimental data.