Optimization of phenolic compounds extraction conditions from lady's bedstraw (Galium verum L.) using historical data design

Lady's bedstraw (Galium verum L.) are often used in the traditional medicine of the Balkan countries. In previous studies it has been shown that the main components of G. verum extracts, phenols and flavonoids are in the form of heterosides with different saccharides. Also, different types of terpenes, which are the main components of essential oil, are present. In this study, we wanted to identify the optimal conditions for the extraction of phenolic compounds from G. verum. For extraction we used methanol, 96% ethanol and 70% ethanol at five time intervals (15, 30, 60, 90 and 120 minutes). Extraction was carried out in conical flasks, on a shaker, at room temperature (25°C). The total phenolic content in the extracts was determined spectrophotometrically, using the standard method with the Folin–Ciocalteu reagent, and the results were expressed as gallic acid equivalents (GAE – mg of gallic acid/g of crude extract). The total phenolic content when 70% ethanol was used were 33.36±1.94, 64.06±1.51, 112.36±3.23, 141.40±3.06 and 142.77±3.28 GAE in 15, 30, 60, 90 and 120 minute respectively. We used historical data design (HDD) in Design Expert 7.0 software to identify optimal extraction conditions. ANOVA analysis showed that there is a statistically significant difference in the amount of extracted phenols in between all time intervals except between 90 and 120 minutes. The results of the optimization analysis showed that the highest yield of total phenols (145.78 GAE) was obtained using 70% ethanol as a solvent in a time of 107.03 minutes (desirability level = 0.996), while the lowest yield was obtained using methanol as a solvent. Equation of model when 70% ethanol is used as a solvent is: Total phenols = 0.26 + 2.30 ∗time + 4.74^−3 ∗ time^2 − 3.70^−5 ∗ time^3. The experimental values agreed with those predicted, thus indicating suitability of the model employed and the success of HDD in optimizing the extraction conditions.