No abstract

Medicinal plants are widely used in folk medicine but quite often their composition and biological effects are hardly known. Our study aimed to analyze the composition, cytotoxicity, antimicrobial, antioxidant activity and cellular migration effects of Anthyllis vulneraria, Fuchsia magellanica, Fuchsia triphylla and Lysimachia nummularia used in the Romanian ethnomedicine for wounds. Liquid chromatography with mass spectrometry (LC-MS/MS) was used to analyze 50% (v/v) ethanolic and aqueous extracts of the plants’ leaves. Antimicrobial activities were estimated with a standard microdilution method. The antioxidant properties were evaluated by validated chemical cell-free and biological cell-based assays. Cytotoxic effects were performed on mouse fibroblasts and human keratinocytes with a plate reader-based method assessing intracellular adenosine triphosphate (ATP), nucleic acid and protein contents and also by a flow cytometer-based assay detecting apoptotic–necrotic cell populations. Cell migration to cover cell-free areas was visualized by time-lapse phase-contrast microscopy using standard culture inserts. Fuchsia species showed the strongest cytotoxicity and the highest antioxidant and antimicrobial activity. However, their ethanolic extracts facilitated cell migration, most probably due to their various phenolic acid, flavonoid and anthocyanin derivatives. Our data might serve as a basis for further animal experiments to explore the complex action of Fuchsia species in wound healing assays.

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.