Quantification of In Vitro Replicative Lifespan Elongation Activity of Hormones, Antioxidants, Plant Extract and Bacterial Exudate by Updated “Overlay Method”

Background/Objectives: Many products that claim to have anti-aging effects have been reported, but their relative potency is not clear. In this study, in vitro replicative lifespan extension (RLE) activity of various groups of physiologically active substances was compared by updated “overlay method”. Methods: Human dermal and periodontal ligament fibroblasts (HDFa, HPLF) were inoculated into the inner 60 wells of 96-well microplate, surround by sterile water to prevent the water evaporation. At Day 1 and Day 8, the cells were overlayed with wide ranges of concentrations (0.01~100 µM) of samples without medium change. Viable cell number was measured by MTT method at Day 15, and then corrected for the variation of cell growth due to the location of inoculated cells. RLE value was calculated as the maximum cell proliferation rate relative to the control. Results: Cell density of HDFa and HPLF at subculture decreased with the passage number, and their growth was stopped at 56 or 85 population doubling levels (PDLs), respectively. Hydrocortisone showed the highest RIE values among six hormones, followed by 3 plant extracts, sodium ascorbate and quercetin. On the other hand, other antioxidants, chlorogenic acid, phenylpropanoids, vanilloids, bacterial products showed little or no RLE effects. However, for HPLF cells, hydrocortisone did not show RLE effects while oxytocin showed slight stimulation. Conclusions: When differences in proliferation due to cell seeding position were corrected, biphasic dose response curve of most of the compounds significantly reduced. The present study suggests the significant role of hormone for the regulation of long-term aging process.