Propolis has gained popularity in recent years as a potential preventive and therapeutic agent due to its numerous health benefits, which include immune system boosting, blood pressure lowering, allergy treatment, and skin disease treatment. The pharmacological activity of propolis is primarily attributed to phenolics and their interactions with other compounds. Given that phenols account for most of propolis's biological activity, various extraction methods are being developed. The resin-wax composition of the propolis matrix necessitates the development of an extraction procedure capable of breaking matrix-phenol bonds while maintaining phenol stability. Therefore, the aim of this study was to assess the stability of two major groups of phenolic compounds, flavonoids and phenolic acids, in propolis methanol/water 50/50 (v/v) extracts obtained after ultrasound-assisted extraction (USE) under different extraction parameters (extraction time and pH) and heat reflux extraction (HRE). The methodology involved varying the USE parameters, including extraction time (5, 10, and 15 min) and pH (2 and 7), followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify phenolic recoveries. Results revealed that benzoic acid and chlorogenic acid derivatives demonstrated excellent stability across all ultrasound extraction procedures. The recoveries of flavonoids were highly diverse, with luteolin, quercitrin, and hesperetin being the most stable. Overall, neutral pH improved flavonoid recovery, whereas phenolic acids remained more stable at pH = 2. The most important optimization parameter was USE time, and it was discovered that 15 min of ultrasound resulted in the best recoveries for most of the phenols tested, implying that phenols bind strongly to the propolis matrix and require ultrasound to break the bond. However, the high variability in phenol extraction and recovery after spiking the propolis sample shows that no single extraction method can produce the highest yield of all phenols tested. As a result, when working with a complex matrix like propolis, the extraction techniques and procedures for each phenol need to be optimized.
Keywords: complex matrix; flavonoids; heat reflux extraction; mass spectrometry; phenolic acids; phenolic recovery.