Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.