Marjoram Extract Modulates Ion Currents, Intracellular Calcium Signaling, and MTT-Reducing Activity in Bovine...

Background/Objectives: Origanum majorana L. has reported antioxidant and other bioactive properties, but its effects on excitability and intracellular Ca²⁺ handling in excitable cells remain poorly defined. We investigated how a hydroalcoholic marjoram extract modulates membrane ion currents, cytosolic Ca²⁺ signaling, and MTT-reducing activity in bovine chromaffin cells and primary rat cortical neurons. Methods: A pressurized liquid O. majorana extract (ethanol:water, 70:30) was chemically characterized by HPLC-PAD. Whole-cell patch-clamp and current-clamp recordings were used to assess voltage-dependent Ca²⁺, Na⁺, and K⁺ currents and membrane excitability in bovine chromaffin cells. Fluo-4-AM measurements were used to evaluate Ca²⁺ responses to K⁺, caffeine, and histamine. MTT assays assessed changes in MTT-reducing activity under veratridine-induced Ca²⁺ overload and oligomycin/rotenone-induced mitochondrial stress in bovine chromaffin cells and cortical neurons prepared from embryonic day 18 Sprague-Dawley rats. For inferential statistics, replicate cells or wells from the same primary culture were averaged and the independent culture was used as the biological unit. Results: Culture-level analysis confirmed significant reductions in voltage-dependent Ca²⁺ and Na⁺ currents at 1.5 µg/mL (1 µL/mL) marjoram extract, together with significant overall effects on the putative Ca²⁺-dependent and voltage-dependent K⁺ current components. The extract depolarized the resting membrane potential and markedly suppressed repetitive action-potential firing. Mean K⁺-evoked Fluo-4 responses were reduced by approximately 20%, although this effect did not reach statistical significance at the culture level. Caffeine-evoked Ca²⁺ responses were higher in the presence of 0.015-1.5 µg/mL (0.01-1 µL/mL) marjoram extract, but these differences did not remain significant after correction for multiple comparisons. Histamine-evoked responses were unchanged. In MTT assays, significant preservation of MTT-reducing activity was limited to selected conditions, mainly at lower extract concentrations in bovine chromaffin cells, whereas the neuronal comparisons did not remain significant after multiplicity correction. Because extract-only, concentration-matched vehicle, cell-free extract/MTT interference controls, and an independent orthogonal assay of cell number or membrane integrity were not included, the MTT findings are reported strictly as changes in MTT-reducing activity and not as direct evidence of cytoprotection or neuroprotection. Conclusions:O. majorana extract modulates membrane excitability through effects on multiple plasma-membrane ion currents. The higher mean caffeine-evoked Ca²⁺ responses are compatible with altered Ca²⁺ mobilization from ryanodine-sensitive intracellular stores, but they do not demonstrate direct activation or sensitization of RyR receptors. The MTT findings do not establish cytoprotection or neuroprotection. These findings are correlative, and a causal contribution of ryanodine-sensitive intracellular stores to the preservation of MTT-reducing cellular metabolic activity remains to be established.