Application of a kosmotrope (Na₂CO₃) and chaotrope (NaCl) in chemometric optimization of aqueous two‐phase extraction of bioactive compounds in <i>Hypoxis iridifolia</i>

Enterolactone, coumaric acid and vitexin are polyphenolic compounds present in a variety of fruits, vegetables, cereals and plants. These bioactive compounds are in high demand due to their antioxidant property in various tissues and organs. The purpose of this study was to develop a simultaneous extraction method, an aqueous two-phase extraction (ATPE) method, that would enable the extraction of these compounds from <i>Hypoxis iridifolia</i>. This environmentally friendly extraction method only applied water and ethanol as extraction solvents for these analytes from the plant matrix. After phase separation, the analytes were salted-out from the aqueous phase into the organic phase with the aid of a chaotrope (NaCl) or kosmotrope (Na₂CO₃). Thereafter, the analytes were withdrawn by a micro-pipette for analysis on the high-performance liquid chromatography-photodiode array detector. Optimization was conducted using a central composite design, where three parameters were examined which involved percentage ethanol, centrifugation time and salt type. Generally, the optimized conditions for extraction were an ethanol percentage of 100% and a centrifugation time of 10 min, which yielded concentrations of 2942, 23,823 and 8881 mg kg^-1 for enterolactone, vitexin and coumaric acid, respectively, in the presence of a kosmotrope. The optimized conditions of extraction in the presence of chaotrope were an ethanol percentage of 66% and a centrifugation time of 10 min with concentrations of 6727, 20,833 and 8618 mg kg^-1 for enterolactone, vitexin and coumaric acid, respectively. The ATPE method involving Na₂CO₃ was a better extractant of all the compounds studied relative to that of NaCl. The superior extraction capability of Na₂CO₃ in ATPE could serve as a prototype for the development of efficient extraction methods to meet the high demand for medicinal compounds derived from natural products.