In order to study the effect of A. mollis rhizome hexane extract against the production of ROS in a HepG2 cell culture, two growing conditions were evaluated: cells treated with the extract under normal culture conditions, and pre-incubation with the extract before oxidative stress was induced with 200 mM H2O2. To this end, 15,000 cells were seeded in each well of a 96-well plate in 200 µL of EMEM supplemented with 2 mM glutamine, 1% non-essential amino acids, 1% FBS, and 1% antibiotics. To carry out this assay, the percentage of serum in the medium was reduced in order to prevent any interaction between the compounds present in the extract and certain components of the serum that could otherwise trigger artifacts with cytotoxic activity [46 (link)].
For the assay under normal culture conditions, after 24 h of incubation, the medium was replaced by various concentrations of the extract or β-sitosterol dissolved in the corresponding culture medium supplemented with 1% FBS. Plates were incubated for a further 24 h at 37 °C in a 5% CO2 atmosphere. A 2-7-dichlorodihydrofluorescein (DCFH-DA) diacetate assay was then performed on the HepG2 cell line to determine intracellular ROS levels [24 (link)]. The measurement of fluorescence was started when the extract or the reference compound was added every 15 min up to a maximum of 90 min. Fluorescence was measured at an excitation wavelength of 485 nm and 529 nm of emission. The results were expressed as a percentage of fluorescence with respect to the control.
For the study under oxidative stress, after 24 h of incubation, the medium was replaced by various concentrations of the extract or β-sitosterol dissolved in the corresponding culture medium supplemented with 1% FBS. After 24 h, the medium was replaced by 0.02 mM DCFH-DA for 30 min. The cells were then washed with PBS, and oxidative stress was induced with 200 mM of hydrogen peroxide. The measurement of fluorescence began when H2O2 was added every 15 min up to a maximum of 90 min. All tests were conducted in triplicate.
For the assay under normal culture conditions, after 24 h of incubation, the medium was replaced by various concentrations of the extract or β-sitosterol dissolved in the corresponding culture medium supplemented with 1% FBS. Plates were incubated for a further 24 h at 37 °C in a 5% CO2 atmosphere. A 2-7-dichlorodihydrofluorescein (DCFH-DA) diacetate assay was then performed on the HepG2 cell line to determine intracellular ROS levels [24 (link)]. The measurement of fluorescence was started when the extract or the reference compound was added every 15 min up to a maximum of 90 min. Fluorescence was measured at an excitation wavelength of 485 nm and 529 nm of emission. The results were expressed as a percentage of fluorescence with respect to the control.
For the study under oxidative stress, after 24 h of incubation, the medium was replaced by various concentrations of the extract or β-sitosterol dissolved in the corresponding culture medium supplemented with 1% FBS. After 24 h, the medium was replaced by 0.02 mM DCFH-DA for 30 min. The cells were then washed with PBS, and oxidative stress was induced with 200 mM of hydrogen peroxide. The measurement of fluorescence began when H2O2 was added every 15 min up to a maximum of 90 min. All tests were conducted in triplicate.
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