Metabolite profiling of propolis polyphenols by microwave-assisted extraction combined with high-performance liquid chromatography using the fused-core technology

The chemical composition of propolis extracts is known to be very complex and influenced by several factors, such as the geographic origin. In propolis from temperate zones, the most representative biologically active compounds are polyphenols, including phenolic acids and flavonoids. Raw propolis can not be used as crude material, but it must be purified by solvent extraction to remove the useless material and preserve the active polyphenol fraction. In this study, a closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of phenolic acids and flavonoids from raw propolis. The optimal extraction conditions were determined by means of response surface experimental design methodology. In comparison with other extraction techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed. The HPLC analyses of propolis extracts were carried out for the first time on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm, Supelco), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by UV/DAD and ion trap mass analyzer. A total of thirty-eight polyphenol compounds were identified in propolis samples, on the basis of the UV, MS and MS2 data. In comparison with a conventional fully porous stationary phase, the fused-core one allowed a good separation of polyphenols and a reduction of both time and solvent usage. The method validation, performed in agreement with ICH guidelines, indicated that the correlation coefficients were > 0.999; the limit of detection (LOD) was in the range 0.5-0.8 ug/mL for phenolic acids and 1.2-3.0 ug/mL for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were found to be ≤ 0.3 and 2.2%, respectively. The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0 mg/g. The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in raw propolis extracts to be used in apitherapy.