Optimization of the solvent extraction of bioactive polyphenolic compounds from aquatic fern Azolla microphylla using response surface methodology

Response surface methodology (RSM) based on a central composite design (CCD) was employed to optimize the experimental conditions for extraction of bioactive polyphenolic compounds from aquatic fern Azolla microphylla. The effects of three independent parameters, namely, methanol concentration (X1:60-85%), extraction temperature (X2:55-80˚C) and extraction time (X3:45-100 min) were investigated to optimize the extraction yields of total phenolic (TPC) and flavonoid (TFC) contents, and compounds showing major antioxidant properties, particularly DPPHsc (2, 2-diphenyl-1-picrylhydrazyl), FRAP (Ferric reducing antioxidant power) and ABTSsc (2, 2’-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt) activities. Data were analyzed by using Design Expert (version 8.0.7.1, Stat-Ease, Inc., Minneapolis, MN, USA) statistical analysis software. The optimum extraction conditions were obtained at methanol concentration (X1): 84.85-85%, extraction temperature (X2): 55˚C and extraction time (X3): 99.76-100 minutes, respectively. Under this condition, the optimum yields of TPC and TFC are 2167.03-2160.44 mg gallic acid equivalents (GAE)/g and 46.11-43.02 mg rutin equivalents (RU)/g of extract. The corresponding antioxidant activities are 80.06-76% DPPHsc, 84-80.54% ABTSsc and FRAP value of 56.65-50.48 μg mol (Fe (II))/g. The experimental results were well matched with the predicted results. Through reversed phase- high performance liquid chromatography (RP-HPLC), rutin and quercetin were identified in the optimally obtained extract of Azolla microphylla. This procedure can be helpful in the food and pharmaceutical industry in studying the optimization of extraction of high quality bioactive products from natural sources.