|Abstrakt:||Calendula officinalis (pot marigold) comes from the Mediterranean region and is a worldwide spread cultural crop. It is a medicinal plant with wide usage in medicine, thanks to its rich representation of natural products. The mutual balance and combination of these substances cause that the marigold has rare healing effects in internal and external use. Significant is its antibacterial, antifungal, anti-viral, anti-inflammatory, antitumor and also its spasmolytic activity. The aim of our diploma thesis was to investigate the influence of selected concentrations (12.5, 25, 50, 100, 200 μg.ml-1) of marigold extract on human ovarian line of stratum granulosum HGL5 cells focusing on secretory activity, viability, presence of proliferation markers and apoptosis. Steroid hormones 17β-estradiol and progesterone were determined by ELISA (Enzyme-linked immunosorbent assay) analysis. After adding marigold extract to HGL5 cells, we observed statistically significant differences in progesterone release. Statistically proven (P≤0,01) stimulation of progesterone secretion by HGL5 cells was recorded in only one experimental concentration of 12,5 μg.ml-1 (0,26 ± 0,03 ng.ml-1) compared to the control group (0,10 ± 0,02 ng.ml-1). In the case of 17β-estradiol, HGL5 cells did not show (P≥0,05) significant changes in its secretion after application of marigold extract. When investigating the viability (AlamarBlue) of HGL5 cells after application of selected concentrations of marigold, we observed a statistically proven reduction at levels of P≤0,01 and P≤0,001 at doses of 100 μg.ml-1 (67,78 ± 0,50) and 200 μg. ml-1 (66,79 ± 0,48%) compared to the control group (71,48 ± 0,99%). The remaining concentrations inconclusively (P≥0,05) reduced the viability of HGL5 cells. Furthermore, we focused in the thesis on the evaluation of proliferation markers (PCNA) and apoptosis (caspase-3) by using of immunocytochemistry and light microscopy. The percentage of cells, which contained the PCNA proliferation peptide PCNA, was relatively balanced in all experimental groups with the addition of marigold extract, only in two concentrations a significant increase was observed. The stimulation of PCNA marker was observed at 25 μg.ml-1 (P≤0,05) with a percentage representation of 51,13 ± 1,38% and at a concentration of 50 μg.ml-1 (P≤0,01) with a value of 54,40 ± 0,85% versus control group that contained 45,11 ± 1,45%. In caspase-3, by adding marigold extract, the presence of the marker increased versus the control depending on used dose. The stimulation of caspase-3 marker was demonstrated at concentration of 100 μg.ml-1 (P≤0,01) with a percentage representation of 44,00 ± 1,85% and at the highest concentration of 200 μg.ml-1 (P≤0,001) with the value of 56,44 ± 1,64%.
The determined results of our thesis indicate a dose-dependent modulation potential of investigated pot marigold extract for the secretory activity, viability, proliferation and apoptosis of human ovarian HGL5 stratum granulosum cells. Our results indicate a potential dose-dependent ability of the pot marigold to engage in regulatory mechanisms of steroidogenesis, proliferation and apoptosis through regulation of progesterone secretion and intracellular markers of PCNA proliferation and caspase-3 apoptosis in HGL5 cells under in vitro conditions|