Optimization of Ultrasonicated Kaempferol Extraction from Ocimum basilicum Using a Box-Behnken Design and Its Densitometric Validation

Kaempferol (KA) is a natural flavonol that can be found in plants and plant-derived foods with a plethora of different pharmacological properties. In the current study, we developed an efficient extraction method for the isolation of KA from ultrasonicated basil leaves (Ocimum basilicum). We successfully employed a Box-Behnken design (BBD) in order to investigate the effect of different extraction variables including methanol concentration (40-80%), extraction temperature (40-60 °C), and extraction time (5-15 min). The quantification of KA yield was carried out by employing a validated densitometric high performance thin layer chromatography in connection with ultraviolet detection (HPTLC-VIS). The obtained data showed that the quadratic polynomial model (R² = 0.98) was the most appropriate. The optimized ultrasonic extraction yielded 94.7 ng/spot of KA when using methanol (79.99%) at 60 °C for 5 min. When using toluene-ethyl acetate-formic acid (70:30:1 v/v/v) as a solvent, KA was detected in basil leaves at an Retention factor (Rf) value of 0.26 at 330 nm. Notably, the analytical method was successfully validated with a linear regression of R² = 0.99, which reflected a good linear relationship. The developed HPTLC-VIS method in this study was precise, accurate, and robust due to the lower obtained results from both the percent relative standard deviation (%RSD) and SEM of the O. basilicum. The antioxidant activity of KA (half maximal inhibitory concentration (IC₅₀) = 0.68 μg/mL) was higher than that of the reference ascorbic acid (IC₅₀ = 0.79 μg/mL) and butylated hydroxytoluene (BHT) (IC₅₀ = 0.88 μg/mL). The development of economical and efficient techniques is very important for the extraction and quantification of important pharmaceutical compounds such as KA.