Design of experiment for extraction optimization and antioxidant assay studies of biologically active compounds of propolis

Abstract

Bees gather propolis from tree sap and combine it with their saliva to seal and sanitize the hive. There are various countries where propolis extracts are used in food and beverage as additives mainly due to the presence of phenolic compounds. Therefore, extraction is the initial and the most vital step in the purification and recovery of phenolic compounds from propolis. The purpose of this study is to enhance propolis extract yield by thoroughly optimizing the extraction process and to evaluate and compare the antioxidant assay of the propolis extracts. Several factors such as the chemical nature of phenolic compounds, composition of solvents, temperature and solvent pH may influence the extraction efficiency and quantity of phenolic compounds. These factors have been extensively studied by univariant optimization; however, the univariant optimization fails to consider the effect of interaction between variables on extraction efficiency. Therefore, the Design of Experiment (DoE) approach is adopted in this study in order to evaluate the correlation between various parameters and their impacts on the extraction of propolis in order to determine TPC, TFC, and DPPH scavenging activity. The DoE was carried out to correctly optimize different extraction parameters including the percentage of ethanol in extraction solvent, temperature and time. The ethanol percentage showed the highest F ratio and minimum p-value for the extraction of 28 total phenolic compounds and flavonoids with a negative correlation showing the presence of more polar compounds in propolis. While in the case of DPPH scavenging assay, the temperature was found to have a significantly positive effect on the extraction efficiency. Optimized extraction parameters obtained by the design were found to be different for TFC, TPC and DPPH, e.g., optimum solvent composition for TFC, TPC and DPPH were 51%, 50% and 90%, respectively. The ultrasonic extracts of propolis showed a higher TPC (574.017 µg GAE/g); in 51% ethanol/water at 25 °C while sonicated for only 2 min. The total flavonoid content (TFC) was found higher (353.16 µg Quercetin/g) in 50% ethanol/water when sonicated for 60 min at 56 °C. DPPH scavenging assay showed higher capacity (1617.27 µg Ascorbic acid/g) in 90% ethanol/water at 62 °C for 60 min sonication. Fourteen propolis samples from Pakistan and Turkey were extracted in order to compare TPC, TFC, and DPPH assay results. In Pakistani propolis, the TPC, TFC, and DPPH scavenging assay varies from 418–1026 µg/g, 264–866 µg/g, and 302–1162 µg/g, respectively. Turkish propolis was demonstrated to have higher TPC, TFC, and DPPH scavenging assay values than Pakistani propolis, with values of 1312–1535 µg/g, 1182–1446 µg/g, and 885–2756 µg/g, respectively. It was found that propolis produced naturally possesses more potential phenolic compounds than propolis purchased from farmer’s markets.