Background: Propolis is multicomponent substance collected by honeybees from various plants. It is known for numerous biological effects and is commonly used as ethanolic extract because most of active substances of propolis are ethanol-soluble. However, water-based propolis extracts could be applied more safely, as this solvent is more biocompatible. On the other hand, water extracts has significantly smaller range and quantity of active compounds. The extraction power of water could be enhanced by adding co-solvent which increases both solubility and penetration of propolis compounds. However, variation of solvents results in different composition of active substances that might have distinct effects. The majority of biological effects of propolis are attributed to the antioxidant properties of its active compounds. Antioxidant effect might be a result of either direct scavenging of ROS or modulation of ROS producing organelle activity. Therefore, the aim of this study was to investigate and compare chemical composition, antioxidant properties and effects on mitochondrial respiration of aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP) and ethanolic (EEP) propolis extracts.
Methods: Chemical composition of propolis extracts was determined using HPLC and Folin-Ciocalteu method. Ability to neutralize H2O2 and intracellular ROS concentration in C6 glioma cells were determined fluorometrically by using 10-acetyl-3,7-dihydroxyphenoxazine and 2',7'-dichlorofluorescein diacetate, respectively. Mitochondrial superoxide generation was assessed under fluorescent microscope by using MitoSOX Red. Oxygen uptake rates of mitochondria were recorded by high-resolution respirometer Oxygraph-2 k.
Results: Our data revealed that phenolic acids and aldehydes make up 40-42% of all extracted and identified compounds in AqEP and Pg-AqEP and only 16% in EEP. All preparations revealed similar antioxidant activity in cell culture medium but Pg-AqEP and EEP demonstrated better mitochondrial superoxide and total intracellular ROS decreasing properties. At higher concentrations, AqEP and EEP inhibited mitochondrial respiration, but Pg-AqEP had concentration-dependent mitochondria-uncoupling effect.
Conclusions: Aqueous and non-aqueous propolis extracts differ by composition, but all of them possess antioxidant properties and neutralize H2O2 in solution at similar efficiency. However, both Pg-AqEP and EEP were more effective in decreasing intracellular and intramitochondrial ROS compared to AqEP. At higher concentrations, these preparations affect mitochondrial functions and change energy production in C6 cells.
Keywords: C6 glioma cell culture; Ethanol; Extract of propolis; Mitochondrial respiration; Polyethylene glycol; Polyphenols; Radical scavenging activity; Superoxide; Water.