The effects of ultrasonic-assisted extraction factors for the main phenolic compound (quercetin) from Hypericum perforatum L. were optimized using the Box–Behnken design (BBD) combined with response surface methodology. The BBD was employed to evaluate the effects of extraction temperature (30–70°C), extraction time (20–80 min), methanol concentration (20–80%, v/v), and HCl concentration (0.8–2.0 M) on the content of one of the major phenolic compounds of quercetin. The extracts were analyzed by high performance liquid chromatography (HPLC). The major phenolic compounds of H. perforatum were isolated and the antioxidant capacity and total phenol content were determined in crude extract and fractions. The optimum conditions were determined as extraction temperature 67°C, extraction time 67 min, methanol concentration 77% (v/v), and HCl concentration 1.2 M. The predicted content of quercetin was 10.81 mg/g dried plant under the optimal conditions and the subsequent verification experiment with 11.09 mg/g dried plant confirmed the validity of the predicted model. The isolated compounds were identified as quercetin, cyanidin, protocatechuic acid, and kaempferol.

Horseradish Peroxidase (HRP) was immobilized by covalent binding onto glutaraldehyde cross-linked chitosan beads and these beads were used for determination of total phenolic content in Prunella L. species. Central composite design (CCD) was employed to optimize the conditions for the maximum HRP activity and to understand the significance and interaction of the factors affecting the activity of immobilized HRP. The results indicated that enzyme concentration and immobilization time were significant factors for the immobilization of HRP. The optimum conditions were determined as enzyme concentration 0.25 mg mL⁻¹, pH 8.0 and immobilization time 20h. The recovered activity was obtained as 81% after immobilization under optimal conditions. Total phenol content was determined in four Prunella L. species (Prunella vulgaris L., Prunella laciniata (L.) L., Prunella orientalis Bornm., Prunella grandiflora L.) extracted using methanol, water and methanol/water (4 : 1, v/v). The enzymatic method is based on the spectrophometric measurement of the final quinone-imine colored product, absorbing at 510 nm, by HRP oxidation in presence of hydrogen peroxide. The results were compared with those obtained by applying the Folin method. The highest total phenol content was obtained with the methanol/water (4 : 1, v/v) extract of Prunella vulgaris L. by immobilized HRP method (63.75 mg gallic acid equivalent (GAE) per g dried plant). Operational stability was determined with immobilized HRP and it indicated that a small enzyme deactivation (15%) occurred after 10 consecutive uses.