COLD PLASMA PRETREATMENT FREQUENCY: EFFECTS ON TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF HIBISCUS SABDARIFFA L. CALYX EXTRACTS

Hibiscus sabdariffa L. calyces (HC) are sources of phenolic compounds with antioxidant capacity and potential for bioingredient production. Cold plasma pretreatment (CPP) may influence the recovery and stability of these compounds and, consequently, extract properties. This study evaluated the effect of CPP frequencies, followed by ultrasound-assisted extraction (UAE), on the total phenolic content (TPC) and antioxidant activity (AA) of HC extracts. AA was assessed using the ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Ground HC (approximately 5 g ) was distributed in Petri dishes and subjected to atmospheric CPP in a parallel-plate configuration at 13 kV and frequencies of 5, 8, and 10 kHz for 20 min. Untreated HC samples served as the control. CPP-treated and control samples underwent UAE with water at a solid-to-liquid ratio of 1:25 (w/v) in an ultrasonic bath (25 kHz; 135 W) for 30 min per cycle, followed by centrifugation (14,700 × g, 4 °C, 10 min). After each cycle, we collected the supernatants and re-extracted the residues under the same conditions until exhaustion, requiring five extraction cycles for CPP-treated samples and six for the control. For each sample, the supernatants from all cycles were pooled and concentrated by rotary evaporation at 36 °C, and the final volume was adjusted to 20 mL. TPC (mg gallic acid equivalents (GAE) g⁻¹ HC), DPPH activity (mg Trolox equivalents (TE) g⁻¹ HC), and FRAP activity (µmol Fe²⁺ equivalents g⁻¹ HC) were determined spectrophotometrically at 765, 515, and 595 nm, respectively. Data were subjected to analysis of variance (ANOVA), followed by Tukey’s test at a 5% significance level, using R software. CPP at 5, 8, and 10 kHz yielded mean TPC values of 9.77 ± 0.51, 9.72 ± 0.17, and 10.33 ± 0.16, respectively, significantly exceeding the control (8.25 ± 0.11; p ≤ 0.05), with increases of 18.5%, 17.8%, and 25.3%. These findings indicate that CPP enhanced phenolic recovery under the extraction conditions. Although treatment at 5 kHz increased TPC, its DPPH activity (13.03 ± 0.77) did not differ significantly from the control (13.82 ± 0.86; p > 0.05). Treatments at 10 and 8 kHz exhibited DPPH activity values of 18.55 ± 1.05 and 17.85 ± 1.96, respectively, representing increases of 34.2% and 29.2% relative to the control (p < 0.05). FRAP activity did not differ significantly among treatments (p > 0.05), ranging from 262.70 ± 19.20 at 8 kHz to 276.60 ± 63.00 in the control. The DPPH and FRAP results suggest that the antioxidant response to CPP depends on the assay used. Thus, CPP at 10 kHz followed by UAE combined the highest mean TPC with significantly greater DPPH activity than the control, indicating potential for producing phenolic-rich HC extracts for bioingredient development.