Screening genotypes and optimizing ultrasonic extraction of phenolic antioxidants from Rheum ribes using response surface methodology

The flowers and stems of Rhubarb (Rheum ribes L.) are known to contain effective antioxidant compounds that have potential antidiarrheal properties in traditional medicine. This study was conducted to screen various genotypes of Rhubarb for their phytochemical and antioxidant activity and optimize the extraction parameters using the response surface methodology (RSM). The study found high diversity among the different genotypes (G1-G13) in terms of their flowers and stems. The total phenolic content (TPC) in the flowers of R. ribes varied significantly, showing values between 9.80 and 81.53 mg GAE g^-1 DW. In the stems, TPC ranged from 2.87 to 16.33 mg GAE g^-1 DW. Similarly, the total flavonoid content (TFC) in the flowers ranged from 0.33 to 1.32 mg Qu g^-1 DW, while in the stems, it was between 0.05 and 0.38 mg Qu g^-1 DW. The antioxidant activity, indicated as µmol Fe²⁺ g^-1 DW, varied from 7.42 to 59.87 in the flowers and from 0.14 to 15.99 in the stems. Hierarchical cluster analysis (HCA) identified five distinct clusters among the collected genotypes. Subsequent analysis of variance and principal components analysis (PCA) revealed that the flowers of G8 (G8F) from Tehran to Lavasan exhibited the highest total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity (FRAP). Given these findings, G8F was chosen for further optimization in the study. The RSM was designed based on a Box-Behnken design (BBD) to determine the optimal extraction conditions, including extraction temperature (30-80 °C), extraction time (5-15 min), and ethanol concentration (25-75%, ethanol to water, v/v). The responses measured were total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, and Ferric reducing/antioxidant power (FRAP) assays. The optimal extraction conditions for all responses or desirability indices were X₁: 80 °C, X₂: 15 min, and X₃: 53.14%, which resulted in TPC (99.32 mg GAE.g^-1 DW), TFC (3.00 mg Qu.g^-1 DW), TAC (1.12 mg cyanidin-3-glucoside (Cy-g) g^-1 DW), FRAP (110.22 µmol Fe⁺²/g DW), and DPPHsc (88.20%). The R² values (0.91-0.99) indicated that the RSM models were acceptable.