Extraction of flavanones from immature Citrus unshiu pomace: process optimization and antioxidant evaluation

Oxidative stress, driven by reactive oxygen and nitrogen species, is implicated in over 100 diseases. Natural antioxidants can mitigate this stress, but measured activities vary with mechanism, structure, and radical type, necessitating multiple assays. Citrus species are rich in flavanone glycosides (hesperidin, narirutin) with diverse bioactivities. Immature Citrus unshiu fruit contains higher flavanone levels than mature fruit, making its pomace a promising source. Previous optimizations largely targeted mature peels and often used methanol (unsuitable for food applications). Ethanol is a GRAS solvent favored for industry. The research question was to develop and optimize an ethanol-based extraction process to maximize recovery of hesperidin and narirutin from ICUP using response surface methodology and to comprehensively evaluate antioxidant activities of the extracts across ten in vitro assays. The study also examined effects of solvent type and number of extraction steps.

Multiple studies have explored citrus flavonoid extraction mainly from mature peels. Lee et al. optimized hesperidin extraction from mature C. unshiu peel (optimal 71.5 °C, 59.0% ethanol, 12.4 h) but required long times at small scale. Li et al. evaluated total phenolics from various citrus peels with ethanol (20–95%) and temperatures up to 80 °C, using single-variable experiments without assessing hesperidin/narirutin composition. Garcia-Castello et al. optimized grapefruit waste extraction (optimum 69 °C, 30% ethanol, 190 min), with relatively low ethanol percentage and long time; temperatures above 70 °C were not tested. Assefa et al. optimized yuzu peel extraction (43.8 °C, 65.5% ethanol, 119.6 min), again with long times and lower temperatures. Safdar et al. used low temperature (40 °C) and long time (20 h) for kinnow peel. Many reports favored methanol for higher yields, but it is unsuitable for food uses. Novel techniques (microwave, ultrasound, subcritical water, supercritical fluids, high pressure) offer advantages but have limited industrial applicability and scalability. There was a gap in optimized methods for immature citrus matrices, specifically ICUP, and in identifying conditions that balance high yield, short time, and GRAS solvents.

Materials and methods included: (1) Sample preparation: Immature C. unshiu fruits from Jeju, Korea were rinsed, quartered, ground, pressed; pomace combined with fine particles, freeze-dried, milled (14–50 mesh), stored at −20 °C. (2) Flavonoid content determination in mature/immature fruits and ICUP: Methanolic extraction (5 g in 30 mL, stirred 30 min, centrifuged; repeated until quantitative recovery), concentrated, volumetrically adjusted, filtered (0.45 μm) for HPLC. Moisture determined at 105 °C. (3) Extraction procedure for ICUP: 1 g dried ICUP extracted with preheated solvent under reflux condenser in shaking water bath (120 rpm) at set conditions; filtrate volume-adjusted; filtered before HPLC. (4) Single-factor experiments: Ethanol concentration 20–100% (v/v); temperature 25–90 °C; solvent-to-feed (S/F) ratio 20–70 mL/g; time 10–60 min. (5) Response surface design: Box–Behnken design with three factors at three levels and 5 center points; factors were temperature (60, 75, 90 °C), ethanol concentration (40, 60, 80%), S/F ratio (20, 30, 40 mL/g), time fixed at 30 min. Second-order polynomial models fitted using DESIGN-EXPERT 11. (6) Number of extractions: Up to three sequential extractions under ethanol-optimal conditions (80.3 °C, 58.4% ethanol, 40 mL/g, 30 min) to assess cumulative yields. (7) Extraction solvents: Compared ethanol, methanol, and acetone under the ethanol-optimized conditions. (8) HPLC analysis: Waters Alliance 2965 with Inertsil ODS-3V (4.6×250 mm, 5 μm); mobile phases: 0.5% acetic acid in water (A) and acetonitrile (B); gradient 15–65% B; flow 1.0 mL/min; detection at 290 nm. Standards verified by retention time and UV spectra. Calibration curves: hesperidin 25–200 mg/mL (R2=0.9996), narirutin 10–80 mg/mL (R2=0.9994). (9) Antioxidant assays (all reported as mg Trolox equivalents per g dry sample): Nitrogen radical scavenging (DPPH at 517 nm; ABTS at 750 nm), RNS scavenging (nitrite via Griess at 540 nm; nitric oxide via modified Griess at 540 nm), ROS scavenging (ORAC fluorescence, hydroxyl radical assay at 536 nm, superoxide anion at 560 nm, hydrogen peroxide at 230 nm), and reducing capacities (reducing power at 700 nm; FRAP at 595 nm). (10) Statistics: Duncan’s multiple range test (p<0.05), Student’s t-test for model validation, Pearson correlations between antioxidant activities and flavonoid contents; analyses via SPSS v24.

• ICUP composition: Immature fruits had 2.32-fold higher hesperidin and 2.34-fold higher narirutin than mature fruits. ICUP hesperidin content matched immature fruit; narirutin was lower in pomace due to transfer to juice (more polar). Quantitatively (μg/g dry sample): immature fruit hesperidin 50,822 ± 662; narirutin 18,359 ± 275; ICUP hesperidin 49,731 ± 808; narirutin 12,969 ± 165.
• Single-factor trends: Optimal ethanol concentration around 60% (yields increased from 20% to 60% ethanol, then decreased). Temperature increased yields up to 75 °C with slight decreases by 90 °C. S/F ratio improved yields up to 40 mL/g; higher ratios gave no significant gain. Optimal extraction times were short (hesperidin ~30 min; narirutin ~20 min), with no significant gains beyond 30 min.
• RSM optimization: Highly significant quadratic models with excellent fit (R2 ≥ 0.989; nonsignificant lack-of-fit). For hesperidin, linear positive effects of temperature and S/F, significant negative quadratic terms; ethanol concentration had a significant negative quadratic effect; interaction terms mostly not significant. For narirutin, similar patterns with a significant interaction between ethanol concentration and S/F.
• Optimal conditions: Joint optimum for both compounds at 80.3 °C, 58.4% ethanol, S/F 40 mL/g, 30 min (desirability 0.977). Predicted yields: hesperidin 66.2% ± 1.1; narirutin 83.7% ± 0.8. Experimental validation matched predictions: hesperidin 66.6% ± 0.9; narirutin 82.3% ± 1.6.
• Number of extractions: Two sequential extractions under optimal conditions recovered most flavanones: first step extracted 67.6% (hesperidin) and 82.4% (narirutin); second step added 24.5% and 14.8%, totaling 92.1% and 97.2%, respectively. A third extraction contributed minimally.
• Solvent comparison (under ethanol-optimized conditions): Hesperidin yields: ethanol 66.6%, methanol 57.3%, acetone 37.7%. Narirutin yields: ethanol 82.3% ≈ methanol 82.5% (not different), acetone 75.1% (lowest). Thus, ethanol outperformed methanol and acetone overall and is GRAS.
• Antioxidant activities (mg TE/g dry sample): Ethanol extract showed highest values across assays compared to methanol and acetone. For ethanol: DPPH 4.4 ± 0.2; ABTS 17.9 ± 0.3; nitrite 17.9 ± 0.6; nitric oxide 4.2 ± 0.1; ORAC 237.5 ± 8.8; hydroxyl radical 206.1 ± 11.3; superoxide anion 394.5 ± 13.3; hydrogen peroxide 74.5 ± 1.7; reducing power 7.8 ± 0.2; FRAP 14.5 ± 0.8. Notably, ROS scavenging activities (ORAC, hydroxyl, superoxide, H2O2) were very high relative to RNS and nitrogen-radical assays.
• Correlations: Antioxidant activities correlated more strongly with hesperidin content than narirutin (e.g., hesperidin correlations 0.923–1.000 for most assays; narirutin 0.741–0.958), except reducing power where the advantage was smaller.
• Process efficiency: Compared to literature on mature peels, this method achieved high yields with short extraction time (30 min), moderate temperature (~80 °C), and a food-grade solvent.

The study addressed the need for an optimized, food-grade process to recover flavanone glycosides from an immature citrus matrix. Single-factor and RSM results showed that moderate ethanol–water mixtures around 58–60% maximize solubility and diffusivity without causing cell dehydration, while elevated temperatures up to about 80 °C enhance solute solubility and decrease solvent viscosity and surface tension, improving mass transfer. An S/F ratio of 40 mL/g was sufficient to achieve near-plateau yields while remaining practical for scale-up. Fixing extraction time at 30 min balanced efficiency and avoided degradation. The optimized conditions produced high single-pass yields and, with a second extraction, achieved near-exhaustive recoveries (≥92% for hesperidin and ≥97% for narirutin), confirming the practical benefit of two-step extraction to overcome equilibrium limits. Ethanol’s superior performance and GRAS status make it preferable to methanol and acetone for food applications. Antioxidant profiling across ten assays demonstrated strong ROS scavenging and reducing capacities, with comparatively lower activity against nitrogen-centered radicals and RNS, highlighting mechanism- and radical-structure-dependent efficacy. Stronger correlations between antioxidant activities and hesperidin content suggest hesperidin is the principal contributor to the observed antioxidant capacity under these conditions. Collectively, these findings validate ICUP as a viable source for flavanone-rich, antioxidant extracts suitable for functional food and nutraceutical use, using an industrially relevant solvent and short processing time.

An ethanol-based extraction of immature Citrus unshiu pomace was optimized using response surface methodology. Optimal conditions were 80.3 °C, 58.4% ethanol, S/F 40 mL/g, 30 min, with two sequential extractions recommended to attain near-complete recovery (hesperidin 92.1%, narirutin 97.2%). Ethanol outperformed methanol and acetone for hesperidin, and matched or exceeded methanol for narirutin, while being a GRAS solvent. The ethanol extract exhibited high ROS scavenging and reducing activities across ten assays, and antioxidant activities correlated more strongly with hesperidin than narirutin content. This process provides a rapid, efficient route to produce antioxidant-rich extracts for functional foods and nutraceuticals. Future work should investigate biological efficacy in cell and animal models (e.g., anti-aging effects) and assess scale-up considerations.

• Antioxidant evaluations were limited to in vitro chemical assays; no cellular or in vivo studies were conducted, and biological efficacy remains to be verified.
• Optimization via RSM fixed extraction time at 30 min; broader time optimization within the multivariate design was not performed.
• Temperature range in RSM was limited to 60–90 °C; behavior above 90 °C was not assessed.
• Solvent comparison applied ethanol-optimized conditions to methanol and acetone; those solvents were not independently optimized, possibly underestimating their best-case performance.
• Study material was ICUP from a single geographic source and season; variability across cultivars, locations, and harvest times was not evaluated.