Application of Ultrasound-Assisted Extraction for Isolating Rhamnetin from Clove (Syzygium aromaticum) Flower Buds: Process Optimization, Identification, and Bioactivity Assessment

The present study aimed to evaluate the effect of ultrasound-assisted extraction (UAE) parameters, specifically extraction time, temperature, and ultrasound amplitude, on extraction yield, bioactive compound content, and antioxidant and cytotoxic activities of ethanolic extracts obtained from flower buds of the clove plant (Syzygium aromaticum). Extraction was performed using 70% ethanol as an environmentally friendly solvent, and the sonication conditions were systematically optimized with clear statistical justification through experimental design. The total phenolic content (TPC) was estimated spectrophotometrically, and the qualitative and quantitative profiles of flavonoids (rhamnetin and isorhamnetin) were analyzed by reversed-phase ultra-high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Rhamnetin and isorhamnetin were selected as specific analytical markers due to their high stability, unique methoxy-substitution patterns, and pivotal role in mediating biological efficacy. DPPH, ABTS, and FRAP assays were conducted to assess the antioxidant properties of the extracts, incorporating pure individual reference standards (gallic acid, rhamnetin, and isorhamnetin) at matching concentrations to account for potential synergistic or antagonistic multi-component interactions. Furthermore, the extract obtained under the optimized extraction conditions was subjected to in vitro evaluation using porcine enterocyte cell lines. The cytotoxic properties of the extract and its effects on cell viability, reactive oxygen species (ROS) generation, and epithelial barrier integrity were assessed, with the latter determined by measuring transepithelial electrical resistance (TEER). UAE parameters significantly influenced extraction efficiency. The optimal extraction conditions, i.e., extraction time: 20 min; temperature: −30 °C; amplitude: 90 µm; power density: 30 W/mL, provided the highest extraction yield (21.80%), which was significantly greater than those obtained using conventional extraction methods (maceration and hydrodistillation). The obtained extract exhibited the highest TPC (320.08 mg gallic acid equivalent/100 g dry matter [DM]) and the highest concentrations of rhamnetin (241.03 ppm) and isorhamnetin (32.80 ppm). Consequently, it showed excellent antioxidant activity, with DPPH radical scavenging activity of 97.28%, FRAP value of 466.27 mg Trolox/100 g DM, and ABTS value of 12.98 µM Trolox/100 g DM. In cell-based in vitro experiments, the highest extract concentrations (≥1000 mg/L) significantly reduced enterocyte viability (p ≤ 0.002), impaired epithelial barrier integrity as determined by TEER (p ≤ 0.005), and induced oxidative stress after 24 h of exposure (p ≤ 0.001). Interestingly, short-term exposure (6 h) to high extract concentrations was associated with decreased intracellular ROS levels (p ≤ 0.05). Lower extract concentrations showed no apparent disruptive effects on intestinal epithelial barrier integrity under the tested conditions. Lower extract concentrations (p ≤ 005) were safe and did not disrupt intestinal epithelial barrier integrity. Thus, UAE with ethanol solvent represents a highly efficient and environmentally friendly alternative to conventional methods for extracting bioactive compounds from clove buds. The obtained extract exhibited a distinct dose- and exposure duration-dependent dual effect, ranging from strong antioxidant activity at low concentrations to cytotoxic effects at high concentrations; this finding highlights the potential utility of this extract in pharmaceuticals and functional foods, provided its dose is carefully regulated.