In order to evaluate the antioxidant effects of the AH and AL flower extracts in vitro, Caco-2 cells were treated for 48 h with extract in aqueous solution (1–20 µg/mL). The dose with the maximum effect observed in the antioxidant reactions described above was chosen. Total RNA was then extracted from 5 × 106 cells following a standard protocol [51 (link),60 (link),61 (link),62 (link),63 (link)]. RNA was quantified spectrophotometrically and 2 µg was back-transcribed using the SuperScript® VILO ™ cDNA synthesis kit (ThermoFisher, Mumbai, India). An aliquot (3.5 µL) of cDNA was then amplified by real-time PCR.
TaqMan® MGB (ThermoFisher) probes were used to evaluate the mRNA expression levels of genes involved in cellular oxidative processes. The probes were heme oxygenase-1 (HO-1) (Hs01110250_m1), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Hs00975961_g1), and NAD(P)H quinone reductase (NQO1) (Hs01045994_m1). The beta-actin gene (Hs01060665_g1) was used for housekeeping. All samples were amplified in triplicate in 96-well optical plates (ThermoFisher) by the ABI Prism 7000 Sequence Detection instrument (Applied Biosystems) following the instrument protocol. Quantification was performed using the comparative method C (T), also called method 2 (−ΔΔCT).
TaqMan® MGB (ThermoFisher) probes were used to evaluate the mRNA expression levels of genes involved in cellular oxidative processes. The probes were heme oxygenase-1 (HO-1) (Hs01110250_m1), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Hs00975961_g1), and NAD(P)H quinone reductase (NQO1) (Hs01045994_m1). The beta-actin gene (Hs01060665_g1) was used for housekeeping. All samples were amplified in triplicate in 96-well optical plates (ThermoFisher) by the ABI Prism 7000 Sequence Detection instrument (Applied Biosystems) following the instrument protocol. Quantification was performed using the comparative method C (T), also called method 2 (−ΔΔCT).
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