Oxidative stress, caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS), contributes to numerous diseases. Natural antioxidants, such as flavonoids found in citrus fruits, offer potential health benefits. While mature citrus peels are frequently studied for flavonoid extraction, immature Citrus unshiu fruit contains higher levels of flavanones, particularly hesperidin and narirutin. Existing flavonoid extraction methods from citrus often utilize methanol, which is unsuitable for food applications. This research aimed to develop an optimized, food-safe ethanol-based extraction method for hesperidin and narirutin from ICUP, maximizing yields and evaluating the antioxidant properties of the resulting extracts using a comprehensive panel of assays. This is crucial for the development of functional food and nutraceutical products rich in these beneficial compounds.

Several studies have investigated flavonoid extraction from mature citrus peels using various methods, including maceration, refluxing, microwave-assisted, ultrasound-assisted, subcritical water, supercritical fluid, and high-pressure extractions. While novel methods often offer advantages in yield, solvent consumption, and extraction time, their industrial applicability remains limited. Maceration and refluxing are common industrial methods, with methanol frequently used as a solvent due to its high extraction efficiency. However, ethanol is preferred for food applications due to its GRAS (Generally Recognized As Safe) status. Previous studies on ethanol extraction of citrus flavonoids have yielded varying results, with differences in optimal conditions depending on factors such as sample type (peel, pulp, or pomace), sample size, and extraction time. Many prior studies used mature citrus peels, while the current study focuses on the underutilized ICUP, which contains higher levels of flavanones. Furthermore, prior studies often utilized a one-variable-at-a-time approach or lacked a comprehensive antioxidant activity evaluation. This study aims to address these limitations by using a comprehensive approach to optimization and testing.

Immature Citrus unshiu Markovich fruits were obtained, processed, and freeze-dried to produce ICUP powder. Single-factor experiments were conducted to evaluate the individual effects of ethanol concentration (20-100%), temperature (25-90 °C), solvent-to-feed (S/F) ratio (20-70 mL/g), and extraction time (10-60 min) on hesperidin and narirutin yields. Response surface methodology (RSM), specifically a Box-Behnken design (BBD), was employed to optimize the extraction process. The number of extractions and the type of solvent (ethanol, methanol, acetone) were also investigated. Hesperidin and narirutin content in the extracts were quantified using high-performance liquid chromatography (HPLC). A total of ten antioxidant assays were performed to comprehensively assess antioxidant activity: DPPH and ABTS radical scavenging assays; nitrite and nitric oxide scavenging assays; ORAC, hydroxyl radical, superoxide anion radical, and hydrogen peroxide scavenging assays; and reducing power and FRAP assays. Statistical analyses, including Duncan's multiple range test, Student's t-test, and Pearson correlation analysis, were conducted using SPSS software.

Single-factor experiments revealed that hesperidin and narirutin yields initially increased with increasing ethanol concentration, temperature, and S/F ratio, before reaching a plateau or decreasing at higher levels. Optimal extraction time was found to be around 30 minutes. RSM optimization using a BBD resulted in optimal extraction conditions of 80.3 °C, 58.4% ethanol, and 40 mL/g S/F ratio, with an extraction time of 30 min. Two sequential extractions were found to be optimal, yielding 92.1% hesperidin and 97.2% narirutin. Ethanol proved to be a more effective solvent than methanol or acetone. The ethanol extract demonstrated high ROS scavenging activity (ORAC, hydroxyl radical, superoxide anion, and hydrogen peroxide), but relatively lower activity against nitrogen radicals and RNS. Correlation analysis indicated a stronger correlation between antioxidant activity and hesperidin content compared to narirutin content.

The optimized extraction method successfully yielded high recoveries of hesperidin and narirutin from ICUP using ethanol, a food-grade solvent. The high ROS scavenging activity of the ethanol extract highlights its potential application in preventing oxidative stress-related diseases. The stronger correlation between antioxidant activity and hesperidin content suggests that hesperidin is a primary contributor to the antioxidant properties of the extract. The study’s findings support the potential of ICUP as a source of antioxidant-rich extracts for functional food and nutraceutical applications. These results contribute to the body of knowledge on citrus waste valorization and the development of natural antioxidant sources.

This study successfully optimized an ethanol-based extraction method for hesperidin and narirutin from ICUP, achieving high yields and demonstrating the potent ROS scavenging activity of the resulting extract. The findings highlight the potential of ICUP as a sustainable source of valuable antioxidant compounds for functional foods and nutraceuticals. Further research could investigate the in vivo effects of these extracts and explore their potential applications in various health-promoting products.

The study focused on in vitro antioxidant activity. Further studies are needed to investigate the in vivo effects of the extracts. The study used a specific cultivar of immature Citrus unshiu; therefore, the results might not be directly generalizable to other citrus varieties or maturity levels. The scope of antioxidant assays, while extensive, does not encompass all potential antioxidant mechanisms or radical species. The study’s results are based on the in-vitro testing and cannot be directly translated to human application without proper clinical trials.