Abstract
Clinopodium tomentosum (Kunth) Govaerts is an endemic species in Ecuador, where it is used as an anti-inflammatory plant to treat respiratory and digestive affections. In this work, effects of a Clinopodium tomentosum ethanolic extract (CTEE), prepared from aerial parts of the plant, were investigated on vascular endothelium functions. In particularly, angiogenesis activity was evaluated, using primary cultures of porcine aortic endothelial cells (pAECs). Cells were cultured for 24 h in the presence of CTEE different concentrations (10, 25, 50, and 100 μg/ml); no viability alterations were found in the 10-50 μg/ml range, while a slight, but significant, proliferative effect was observed at the highest dose. In addition, treatment with CTEE was able to rescue LPS-induced injury in terms of cell viability. The CTEE ability to affect angiogenesis was evaluated by scratch test analysis and by an in vitro capillary-like network assay. Treatment with 25-50 μg/ml of extract caused a significant increase in pAEC's migration and tube formation capabilities compared to untreated cells, as results from the increased master junctions' number. On the other hand, CTEE at 100 μg/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25 μg/ml. The CTEE phytochemical composition was assessed through HPLC-DAD; rosmarinic acid among phenolic acids and hesperidin among flavonoids were found as major phenolic components. Total phenolic content and total flavonoid content assays showed that flavonoids are the most abundant class of polyphenols. The CTEE antioxidant activity was also showed by means of the DPPH and ORAC assays. Results indicate that CTEE possesses an angiogenic capacity in a dose-dependent manner; this represents an initial step in elucidating the mechanism of the therapeutic use of the plant.
Highlights
Medicinal plants are presently in demand, and their acceptance is increasing progressively
Human Endothelial Serum Free Medium, heat-inactivated fetal bovine serum (FBS), antibiotic-antimycotic (100x solution), Dulbecco’s phosphate buffered saline (DPBS), and phosphate buffered saline (PBS) were purchased from Gibco Life Technologies (Carlsbad CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); Folin-Ciocalteu’s phenol reagent; 1,1-diphenyl-2-picrylhydrazyl (DPPH); 6-hydroxyl2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox); pure standards of phenolic acids (4-hydroxybenzoic, gallic, caffeic, chlorogenic, ferulic, p-coumaric, synapic, syringic, trans-cinnamic, and rosmarinic acids); flavonoids; and HPLC-grade solvents were purchased from Sigma-Aldrich Italia (Milan, Italy)
MTT tests were carried out to evaluate if Clinopodium tomentosum ethanolic extract (CTEE) affects porcine aortic endothelial cells (pAECs) viability in control conditions or during an LPS-induced damage
Summary
Medicinal plants are presently in demand, and their acceptance is increasing progressively. Bernardini et al [30] have isolated and characterized a primary culture of porcine aortic endothelial cells (pAECs), which have been already used as an in vitro model to evaluate different substances and plant extract effects on the endothelial functions [31,32,33,34]. The effect of CTEE on LPS-induced injury was studied; pAECs were seeded in 96-well culture plates at a density of 12 × 103 cells/well and incubated for 24 h, exposed to different concentrations of CTEE (10, 25, 50, and 100 μg/ml) in the presence of LPS (25 μg/ml).
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