Optimization of phenolics extraction and isolation from gymnosperms growing in Egypt using response surface methodology: <i>Dioon edule</i> Lindl leaves as a case study

Gymnosperms are recognized as rich sources of phenolic constituents, particularly biflavonoids, which exhibit a wide range of biological activities, including antioxidant, anti-inflammatory, and anticancer effects. However, conventional extraction techniques such as maceration and Soxhlet extraction are often limited by prolonged extraction times and high solvent consumption. In this study, traditional extraction methods were systematically compared with modern techniques, namely microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE), for the recovery of phenolic compounds from the leaves of <i>Dioon edule</i> Lindl. (<i>D. edule</i>) cultivated in Egypt. Among the evaluated methods, UAE afforded the highest total phenolic content (11.28 mg gallic acid equivalents GAE g^-1 dry plant matter (DM)), total flavonoid content (8.56 mg rutin equivalents RE g^-1 DM), and antioxidant activity (37.1% DPPH radical scavenging activity, IC₅₀ = 151.25 µg mL^-1). Based on these findings, the UAE was further optimized using response surface methodology (RSM), considering temperature, extraction time, and solvent-to-plant material ratio as critical variables. Under the optimized conditions (80 °C, 100 min, 50 : 1 mL g^-1), the extraction yield increased by 27%, while phenolic and flavonoid contents increased by 15% and 33%, respectively. Antioxidant activity was significantly enhanced, with DPPH inhibition increasing to 58.2% and IC₅₀ decreasing to 75.6 µg mL^-1. In addition, antibacterial and cytotoxic activity assays consistently supported the superiority of UAE. Chromatographic separation of the optimized UAE extract led to the isolation of five bioactive biflavonoids, identified as 7,4',7',4‴-tetra-<i>O</i>-methylamentoflavone (1, isolated from dichloromethane fraction (DCM)) and total methanolic extract), isoginkgetin (2, DCM fraction), sciadopitysin (3, ethyl acetate (EtOAc) fraction), amentoflavone (4, EtOAc fraction), and kayaflavone (5, total methanolic fraction). Structural elucidation was achieved through comprehensive spectroscopic analyses and comparison with reported data. These compounds were obtained in higher yields compared with previous reports based on conventional extraction methods. Therefore, the study provides a systematic evaluation and statistical optimization of UAE for phenolic recovery from <i>D. edule</i>, coupled with phytochemical profiling and preliminary biological assessment. The findings indicate that optimized UAE conditions enhance extraction efficiency and phenolic recovery relative to the tested conventional approaches.