Phytochemicals Rosmarinic Acid, Ampelopsin, and Amorfrutin-A Can Modulate Age-Related Phenotype of Serially Passaged Human Skin Fibroblasts in vitro

Abstract

One of the aims of the EU-funded Research and Innovation Action (RIA), titled “Ageing with Elegans” (AwE) is to enhance better understanding of the factors causing health and disease in aging and develop evidence-based preventive, diagnostic, therapeutic, and other strategies. The work package-5 of this project is focused on testing the effects of phytochemicals of natural and synthetic origin on aging, longevity, and health of human cells in vitro , after the initial screening using the animal model systems of nematodes and rats and mice. Accordingly, the first series of three compounds, rosmarinic acid (ROSM), ampelopsin (AMPEL), and amorfrutin-A (AMOR), were selected to test for their short-term and long-term effects on human skin fibroblasts undergoing aging and senescence in vitro. The lifelong modulatory effects of these compounds were tested individually at two doses (0.5 and 1.0 μM), selected after a short-term dose response check of a 20,000-fold range (0.01–200 μM). The results show that these compounds do have some beneficial effects in terms of supporting the long-term lifelong growth and enhanced stress tolerance of serially passaged cells. These effects seem to be achieved by reducing the extent of loss of telomeres, of 5-methyl-cytosine (5-mC) and of 5-hydroxymethyl-cytosine (5-hmC), by reducing the accumulation of oxidative DNA damage product 8-OHdG. There is also some indication that these compounds induce at least one of the stress responses in terms of the increased synthesis of heat shock protein Hsp70. Thus, these phytochemicals may be potential hormetins, which bring about their health beneficial effects by the phenomenon of mild stress-induced hormesis.

Plant-Derived Molecules α-Boswellic Acid Acetate, Praeruptorin-A, and Salvianolic Acid-B Have Age-Related Differential Effects in Young and Senescent Human Fibroblasts In Vitro

“…The stock solutions were stored at 4 • C until further use when appropriate volume from the individual stock solution was added to the complete culture medium in the flasks having the cells. The cell culture medium was replaced with fresh medium with or without test compounds on a weekly basis [13]. As described previously, there were no detectable effects of DMSO on cell survival, growth, and metabolic activity at the doses used in the present studies [13].…”

“…The cell culture medium was replaced with fresh medium with or without test compounds on a weekly basis [13]. As described previously, there were no detectable effects of DMSO on cell survival, growth, and metabolic activity at the doses used in the present studies [13].…”

“…Test compounds ABC, PTA, SAB were dissolved individually in DMSO making a stock solution of 1 mM each, having a maximum concentration of DMSO at 0.5% [13]. The stock solutions were stored at 4 • C until further use when appropriate volume from the individual stock solution was added to the complete culture medium in the flasks having the cells.…”

“…These cells were established from a skin biopsy from the abdomen region of a healthy 38-year-old Caucasian woman. Method for PCS cell culturing and exposure to different test compounds is described previously [13]. Briefly, PCS cells were thawed from a frozen ampoule at passage 10 (p10), and were grown in plastic tissue culture flasks with growth area 75 cm 2 in an incubator at 37 • C, 5% CO 2 , 95% humidity, and atmospheric oxygen conditions in Dulbecco's Modified Eagle's Medium (DMEM; Lonza, Walkersville, MD, USA).…”

“…Early passage cells up to p14 were considered as young, late passage cells at p51 were considered as near-senescent, and ultimate p58 cells were considered as fully senescent. Cell numbers were monitored by using a Coulter counter, and the microscopic-photographic records of cells were kept as phase-contrast pictures either of live cells or of methanol-fixed and Giemsa stained cells, using an inverted Zeiss microscope [13].…”