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

Dietary guidelines recommend the consumption of flavonoid-rich extracts for several health benefits. Although immature Citrus unshiu pomace (ICUP) contains high levels of flavanone glycosides, many studies have concentrated on the optimization of flavonoid extraction from mature citrus peels. Therefore, we developed an optimized extraction method for hesperidin and narirutin from ICUP, and evaluated their antioxidant activities using ten different assay methods. The extraction conditions for the highest flavonoid yields based on a response surface methodology were 80.3 °C, 58.4% (ethanol concentration), 40 mL/g (solvent/feed), and 30 min, where the hesperidin and narirutin yields were 66.6% and 82.3%, respectively. The number of extractions was also optimized as two extraction steps, where the hesperidin and narirutin yields were 92.1% and 97.2%, respectively. Ethanol was more effective than methanol and acetone. The ethanol extract showed high scavenging activities against reactive oxygen species but relatively low scavenging activities for nitrogen radicals and reactive nitrogen species. The antioxidant activities showed a higher correlation with hesperidin content than narirutin content in the extracts. This study confirms the potential of an optimized method for producing antioxidant-rich extracts for the functional food and nutraceutical industries.

Extraction procedure of immature C. unshiu pomace. The dried ICUP (1 g) was transferred to a flask. The preheated extraction solvent was added to the sample, and then a condenser (glass tube, 6.2 mm ID × 400 mm length) was connected to the flask to collect a vaporized solvent. The extraction was performed in a shaking water bath (120 rpm) at the desired experimental conditions. The extraction mixture was filtrated and adjusted to a specific volume with an extraction solvent. All extracts were filtrated using a syringe filter (0.45 μm) before HPLC analyses.

Number of extractions.
To determine the effect of the number of extractions on the yields of hesperidin and narirutin from ICUP, extractions were done three times under the optimum conditions for ethanol (80.3 °C, 58.4%, and 40 mL/g dry sample for 30 min).
Extraction solvents. The effects of different extraction solvents on the yields of hesperidin and narirutin from ICUP were evaluated using methanol and acetone under the optimum conditions obtained using ethanol (80.3 °C, 58.4%, and 40 mL/g dry sample for 30 min).

HPLC analyses.
The contents of hesperidin and narirutin in the extracts were quantified as described by Kim and Lim 25 . The hesperidin and narirutin profiles in the extracts were analyzed using Alliance 2965 HPLC (Waters Corp., Milford, MA, USA). An Inertsil ODS-3 V column (4.6 mm × 250 mm, 5 μm particle size, GL www.nature.com/scientificreports/ Science, Tokyo, Japan) was used for the separation of each flavonoid. Acetic acid (0.5%) in water (phase A) and acetonitrile (phase B) were utilized for the mobile phase. The solvent flow rate was 1.0 mL/min, using gradients of B: 0 min 15%, 8 min 25%, 15 min 25%, 35 min 65%, 37 min 65%, and 39 min 15%. The hesperidin and narirutin were detected at 290 nm. Each flavonoid was identified by comparing with its retention time and UV-visible spectrum to those of the standard compound. Calibration curves were constructed between absorbance at 290 nm and the concentrations of hesperidin (25-200 mg/mL) and narirutin (10-80 mg/mL) in methanol; the correlation coefficients (R 2 ) were 0.9996 and 0.9994, respectively.
Antioxidant activity. Nitrogen radical scavenging activities (DPPH and ABTS) were determined. DPPH radical scavenging activity was evaluated as described by Ye et al. 26 . Each extract (0.1 mL) was mixed with 2.0 mL of a DPPH solution (0.2 mM) and left for 30 min. The absorbance was determined at 517 nm. ABTS radical scavenging activity was also evaluated as described by Yi et al. 27 . Each extract (0.02 mL) was mixed with 0.98 mL of an ABTS solution (0.2 mM) and left for 30 min at 30 °C. The absorbance was evaluated at 750 nm. RNS scavenging activities (nitrite and nitric oxide) were measured. Nitrite scavenging activity was evaluated as described by Kim and Lim 25 . Each extract (0.2 mL) was mixed with 0.1 mL of sodium nitrite (1 mM) and 0.7 mL of HCl (0.1 N) and left for 60 min at 37 °C. Then, added with acetic acid (0.5 mL) and Griess reagent (0.4 mL) and left for 15 min. The absorbance was determined at 540 nm. Nitric oxide radical scavenging activity was also evaluated as described by Soares et al. 28 . Each extract (0.25 mL) was mixed with 0.5 mL of sodium nitroprusside (10 mM) and left for 3 h at 25 °C under the light. Then, added with 0.75 mL of modified Griess reagent (2% sulphanilamide + 0.2% N-(1-naphthyl) ethylenediamine dihydrochloride in 5% phosphoric acid). The absorbance was measured at 540 nm.
ROS scavenging activities (ORAC, hydroxyl radical, superoxide anion radical, hydrogen peroxide) were determined. ORAC was measured as the methods described by Ye et al. 26 and Kim and Lim 25 . Each extract (0.025 mL) was mixed with 0.15 mL of fluorescein sodium salt solution (78 nM), left for 15 min at 37 °C, and added with 0.025 mL of AAPH (250 mM). The fluorescence was determined at 37 °C every 3 min for 2 h (excitation, 485 nm; emission, 535 nm). Hydroxyl radical scavenging activity was measured as the method described by Sannasimuthu et al. 29 . Each extract (0.2 mL) was mixed with 0.2 mL of 1,10-phenanthroline (5 mM), 0.2 mL of EDTA (15 mM), and 0.2 mL of FeSO 4 (5 mM). The reaction was started with an addition of 0.2 mL of H 2 O 2 (0.03%) at 37 °C for 60 min. The absorbance was evaluated at 536 nm. Superoxide anion radical scavenging activity was measured as described by Kuda et al. 30 . Each extract (0.1 mL) was mixed with 0.1 mL of (0.156 mM) and 1 mL of (0.468 mM) NADH. Then, added with 0.1 mL of phenazine methosulphate (0.06 mM) and left for 5 min. The absorbance was evaluated at 560 nm. Hydrogen peroxide scavenging activity was evaluated as described by Oh and Shahidi 31 . Each extract (0.4 mL) was mixed with 0.6 mL of H 2 O 2 (40 mM) and left for 40 min at 30 °C. The absorbance was evaluated at 230 nm.
Reducing abilities (reducing power and FRAP) were determined. Reducing power was measured as described by Kim and Lim 25 . Each extract (0.1 mL) was mixed with 0.5 mL of phosphate buffer (0.2 M) and 0.5 mL of potassium ferricyanide (0.1%) and left for 20 min at 50 °C. Then, added with 0.5 mL of trichloroacetic acid (10%) and 0.5 mL of ferric chloride (0.1%) and left for 5 min. The absorbance was determined at 700 nm. Ferric reducing antioxidant power (FRAP) was evaluated as the method described by Ye et al. 26 . The FRAP solution was prepared with 300 mM acetate buffer, 20 mM FeCl 3 , and 10 mM TPTZ (1:1:10 (v/v)). FRAP reagent (3 mL) and distilled water (0.3 mL) were added to 0.2 mL of extract and left for 30 min at 37 °C. The absorbance was observed at 595 nm. All antioxidant activities were expressed as mg Trolox equivalents (mg TE)/g dry sample.
Statistical analyses. The differences between experimental data were analyzed using Duncan's multiple range test (p < 0.05). The validity between the predicted and experimental data was analyzed using the Student's t-test. Pearson correlation coefficients between antioxidant activities and flavonoid contents in the extracts were also calculated using a bivariate correlation analysis. All statistical analyses were performed using SPSS software (ver. 24.0; SPSS Inc., Chicago, IL, USA).

Results and discussion
Flavonoid compositions of mature and immature C. unshiu fruits and pomace. The flavonoid compositions of mature and immature C. unshiu fruits and pomace were determined ( Table 2). Hesperidin and narirutin are the major flavonoids (flavanone glycosides), and sinensetin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and tangeretin are the minor flavonoids (polymethoxylated flavones) in C. unshiu. The contents of hesperidin and narirutin were 2.32-and 2.34-fold higher in immature than mature C. unshiu fruits, respectively. Therefore, immature C. unshiu fruits may be a good candidate as a flavonoid supplement for several health benefits. The content of hesperidin in immature C. unshiu pomace was the same as that in immature fruits, but that of narirutin in immature pomace was lower than that in immature fruits because it was transferred to the juice when the fruits was squeezed using an extractor due to the more polar property of narirutin compared with hesperidin 32 .
Analyses of single-factor effect experiments. The effects of extraction parameters (ethanol concentration, temperature, S/F ratio, and extraction time) on the extraction yields of hesperidin and narirutin from ICUP were evaluated individually. The extraction yield (%) was defined as the mass (mg) of each flavonoid in the extract divided by that (mg) in the raw ICUP (Table 2). Figure 1a shows the effects of ethanol concentration (20-100% (v/v)) on the extraction yields of hesperidin and narirutin at 60 °C, 30 mL/g dry sample, and 30 min. The yields of hesperidin and narirutin increased with increasing ethanol concentration from 20 to 60%, but decreased thereafter. Therefore, 40%, 60%, and 80% ethanol were chosen as the RSM working ranges. Generally, Scientific Reports | (2020) 10:19950 | https://doi.org/10.1038/s41598-020-76965-8 www.nature.com/scientificreports/ the mixture of ethanol and water increases the extraction yields of flavonoids due to the decrease in dielectric constant of the solvent and the increases of solubility and diffusivity of the solute 16,33 . Relatively consistent with this result, a few studies have reported that an approximately 60% ethanol-water mixture is appropriate for phenolic extraction from C. unshiu peel 21 and yuzu (C. junos Sieb ex Tanaka) peel 17 . Xu et al. 34 also reported that the solubility of pure hesperidin in 60% ethanol solution was more than fourfold higher than that in 20% ethanol solution at 60 °C. However, high concentrations of ethanol do not aid extraction due to the dehydration and collapse of plant cells and the denaturation of cell wall proteins 16 . The effects of temperature (25-90 °C) on the extraction yields of hesperidin and narirutin were measured at 60%, 30 mL/g dry sample, and 30 min (Fig. 1b). The hesperidin yield significantly increased with an increase in temperature from 25 to 75 °C, but slightly decreased at 90 °C. Narirutin showed almost the same trend as hesperidin. Therefore, 60 °C, 75 °C, and 90 °C were chosen as the RSM working ranges. Generally, high temperature increases the extraction yields of flavonoids due to increasing solubility 19 but decreasing viscosity and surface tension of the solvent 35 . The solubility of pure hesperidin in 60% ethanol-water solution was 1.6-fold higher at 60 °C than at 25 °C 34 . The viscosity of the 63% ethanol-water mixture was 2.313 mPa s at 25 °C, but decreased to 1.108 mPa s at 50°C 36 . However, phenolic compounds can be decomposed and degraded by high temperatures 14 .
The effects of S/F ratio (20-70 g/mL) on the extraction yields of hesperidin and narirutin were also investigated at 60%, 60 °C, and 30 min (Fig. 1c). The yields of both increased with increasing S/F ratio from 20 to 40 mL/g dry sample, after which there were no significantly different effects on the yields (p < 0.05). Generally, high S/F ratio increases the extraction yields of flavonoids due to the increase in concentration gradient between the sample and extraction solvent, resulting in increased mass transfer 17,23 . However, a high S/F ratio is economically inefficient due to high-energy consumption in later concentration steps. Therefore, it is important to select the appropriate range of S/F ratio when optimizing the extraction conditions 17,19 . Hence, 20, 30, and 40 mL/g dry sample were chosen as the RSM working ranges.
The effects of extraction time (10-60 min) on the extraction yields of hesperidin and narirutin were determined at 60%, 60 °C, and 30 mL/g dry sample (Fig. 1d). The extraction yields of hesperidin and narirutin were highest at 30 and 20 min, respectively, and there were no significantly different effects on the yields (p < 0.05) thereafter. This can also be explained by Fick's second law of diffusion, which predicts the final equilibrium of the solute concentration between the sample matrix and extraction solvent after a certain period of time 35 . Because an extraction time longer than 30 min is not required for hesperidin or narirutin, the extraction time was fixed at 30 min throughout the optimization process. Iglesias-Carres et al. 19 reported that an extraction time longer than 30 min did not produce any significant increases or decreases in total phenolic content of the aqueous methanol extract from sweet orange pulp. However, the extraction of hesperidin from C. unshiu peel at 1 g/10 mL, 71.5 °C, and 59.0% ethanol needs an extraction time of 12.4 h 21 , and the extraction of hesperidin and naringin from yuzu peel at 1 g/37.1 mL, 43.8 °C, and 65.5% ethanol requires 120 min 17 . Therefore, the optimal conditions for recovering phenolic compounds from citrus may depend on the citrus matrix (peel, pulp, or pomace) used.

Analyses of RSM experiments
Response surface optimization. The extraction parameters (extraction temperature, ethanol concentration, and S/F ratio) for the highest yields of hesperidin and narirutin from ICUP were optimized by RSM. Their extraction yields are shown in Table 3, and the results of analysis of variance (ANOVA) of the regression models are presented in Table 4. The regression models for both compounds were a good fit with the experimental data, with low p-values (p < 0.05), high R 2 values (R 2 ≥ 0.989), nonsignificant lack-of-fit (p > 0.05), and low coefficients of variance (CV ≤ 5%) 37 . The following second-order polynomial equations can be used to estimate the optimal conditions for maximizing each flavonoid:  www.nature.com/scientificreports/ www.nature.com/scientificreports/ where X 1 , X 2 , and X 3 are the test variables (temperature, ethanol concentration, and S/F ratio, respectively). The regression coefficient for each term in Table 4 indicated the effects of three variables on the extraction yields. The response surface plots also facilitated the visualization of the significance of each extraction variable on the yields (Fig. 2). The positive coefficients of the linear terms (X 1 and X 3 ) and the negative coefficients of the quadratic terms (X 1 2 and X 3 2 ) with significant p-values (p < 0.05) indicated that the yields increased with increases in temperature and S/F ratio, peaking, and then no longer increased with increasing temperature and S/F ratio. A few studies have reported that temperature has positive linear and negative quadratic effects on the optimization of extraction of phenolics from C. unshiu peel 21 and yuzu (C. junos Sieb ex Tanaka) peels 17 . On the other hand, the nonsignificant positive linear term (X 2 ) and significant negative quadratic term (X 2 2 ) of ethanol Table 3. Box-Behnken design and corresponding hesperidin and narirutin yields from immature C. unshiu pomace. The extraction yield (%) was calculated as the mass (mg) of each flavonoid in the extract divided by that (mg) in the raw immature C. unshiu pomace.
Run no.  www.nature.com/scientificreports/ concentration indicated that the extraction yield curves of both compounds took the form of a bisymmetry quadratic function with increasing ethanol concentration, and there were maximum ethanol concentrations in both extractions, after which they started to decrease 17,18 . Only the interaction term (X 2 X 3 ) between ethanol concentration and S/F ratio for narirutin was significant (p < 0.05), indicating that those two parameters significantly affected the yield each other. Based on the optimized regression models, the optimal extraction conditions at the highest desirability (1.0) were 81.5 °C, 58.4%, and 39.6 mL/g dry sample for hesperidin; and 78.8 °C, 58.4%, and 40.0 mL/g dry sample for narirutin ( Table 5). The optimum temperature of hesperidin was 2.7 °C higher than that of narirutin, because hesperidin has a lower solubility in water than narirutin 32 . The optimal extraction conditions at the highest desirability (> 0.977) for simultaneous extraction of hesperidin and narirutin were 80.3 °C, 58.4%, and 40.0 mL/g dry sample, which gave the predicted maximum yields of 66.2% and 83.7% for hesperidin and narirutin, respectively.  Table 6). The yields of hesperidin and narirutin at these conditions were 66.6 ± 0.9% and 82.3 ± 1.6%, respectively, in good agreement with the predicted values by the model equations at p < 0.05. Therefore, the response models accurately predicted the extraction yields of hesperidin and narirutin within the tested ranges of extraction parameters. Therefore, the temperature, ethanol concentration, and S/F ratio were 80.3 °C, 58.4%, and 40 mL/g dry sample for the rest of the study.

Number of extractions.
The effects of the number of extractions were evaluated on the recoveries of each compound from ICUP (Fig. 3). Considerable amounts of hesperidin (67.6%) and narirutin (82.4%) were extracted from the first extraction step and then 24.5% and 14.8% were obtained from the second step, respectively. Because most hesperidin (92.1%) and narirutin (97.2%) were extracted from ICUP in the first two extraction steps and considering practical and economic points, two sequential extractions were proposed as the optimized number of extractions. Iglesias-Carres et al. 19 reported that 63% and 24% of hesperidin was extracted from C. sinensis pulp in the first and second extraction steps at the optimum conditions (90% methanol, 55 °C, and 20 mL/g dry sample), respectively.
Effect of extraction solvent. To compare the effects of different solvents commonly used for polyphenol extraction on the yields of hesperidin and narirutin, ICUP was extracted using methanol or acetone under the optimized conditions obtained using ethanol (80.3 °C, 58.4%, and 40 mL/g dry sample for 30 min) (Fig. 4). The yield of hesperidin was highest at 66.6% in ethanol, followed by 57.3% in methanol and 37.7% in acetone. The extraction yield of narirutin in ethanol (82.3%) was statistically the same as that in methanol (82.5%), and that in acetone was the lowest (75.1%). Ethanol was more effective for extracting hesperidin and narirutin than metha- Table 5. Optimized extraction conditions and predicted yields of hesperidin and narirutin. S/F ratio: solvent to feed ratio (mL/g dry sample). Data are the mean ± SD (n = 3).  Table 6. Predicted and experimental yields of hesperidin and narirutin in the optimum conditions. S/F ratio: solvent to feed ratio (mL/g dry sample). The mean values with the same letter ( a ) in each row are not significantly different (p < 0.05 by Student's t-test).  www.nature.com/scientificreports/ nol and acetone. Generally, ethanol is a suitable extraction solvent for flavonoids and their glycosides, catechol, and tannin; methanol for phenolic acid and catechin; and acetone for high-molecular-weight polyphenols such as proanthocyanidins and tannins 35 .
In some other studies, higher extraction yields of hesperidin were obtained using methanol than ethanol. Magwaza et al. 38 reported that the yield of hesperidin from mandarin (C. reticulata) rinds was higher in 70% methanol than in 80% ethanol at 35 °C. Iglesias-Carres et al. 19 also observed that the hesperidin yield from C. sinensis pulp was higher in 90% methanol than in 90% ethanol at 55 °C. These differences are thought to be due to differences in the extraction temperature and ethanol concentration. In most extraction studies using ethanol as the solvent, the optimal extraction temperature was 48-95 °C, and the optimal ethanol concentration was 51-59% for phenolic and flavonoid compounds from kaffir lime peels 39 , C. unshiu peels 21 , and yuzu peels 17 .
Antioxidant activity. The antioxidant activities of natural antioxidants from plant materials depend on the reaction mechanism based on the multiplicity and heterogeneity of the matrix and the distribution of antioxidant compounds between the lipophilic and hydrophilic phases. Therefore, the antioxidant activities of plant extracts cannot be properly assessed by only one method 40,41 . In this work, 10 assay methods were used to comprehensively evaluate the antioxidant activities of ethanol, methanol, and acetone extracts: radical scavenging activities (DPPH, ABTS, nitric oxide, hydroxyl, superoxide anion, and ORAC), scavenging activities of nitrite and hydrogen peroxide, and reducing capacity (FRAP and reducing power) ( Table 7).
DPPH and ABTS radical scavenging activities are commonly used to measure the antioxidant activities of natural antioxidants due to the stability of nitrogen radicals and the simple method used to measure these 42 . DPPH assays use radicals dissolved in organic solvents, which is applicable to the hydrophobic system, but the ABTS assay is applicable to both hydrophilic and lipophilic systems 43 . DPPH and ABTS radical scavenging activities were higher in the ethanol extract than in methanol and acetone extracts. The ABTS radical scavenging activity was 4.06-fold higher than the DPPH radical scavenging activity in the ethanol extract, indicating that its radical scavenging activity works better in a hydrophilic system. RNS and ROS are directly involved in oxidative stress and are closely correlated with the development of several human diseases including atherosclerosis, diabetes, chronic inflammation, and neurodegenerative disorders 3 . Nitrite and nitric oxide radicals are representative RNS. RNS scavenging activity was higher in the ethanol extract than in methanol and acetone extracts. The nitrite scavenging activity was 4.26-fold higher than nitric oxide radical scavenging activity in the ethanol extract. ROS scavenging activities (ORAC, hydroxyl radical,   44,45 . Therefore, the strong and rapid ROS scavenging properties of the ethanol extract can actually prevent the oxidation of food and help fight human diseases caused by ROS. The reducing capacity of each extract was also measured using two methods: a reducing power assay and an FRAP assay. Both methods are commonly used to measure the reducing power of natural antioxidants 45 . The reducing power and FRAP were higher in the ethanol extract than in the methanol and acetone extracts. The FRAP was about 1.85-fold higher than reducing power in the ethanol extract. This difference between two methods may be due to their different reducing mechanisms. In FRAP, the antioxidant reduces Fe 3+ to Fe 2+ in the Fe-TPTZ complex 46 , whereas in reducing power, the antioxidant reduces ferricyanide to ferrocyanide 47 . The ethanol extract showed very high scavenging activities against the radicals containing only oxygen (ROO• and •O 2 − ) or oxygen with hydrogen (•OH and H 2 O 2 ), but relatively low scavenging activities against nitrogen radicals (DPPH• and ABTS•) and nitrogen containing radicals (NO 2 − and NO•), and relatively low reducing activities against the Fe-complex containing nitrogen (Fe(CN) 6 3− and [Fe(III)(TPTZ) 2 ] 3+ ). These results indicate that the antioxidant activity of the extract from ICUP may vary depending on the structure of the radicals, the reaction mechanisms, and the flavonoid types. Table 8 shows the Pearson correlation coefficients representing the relationship between antioxidant activities and the contents of hesperidin and narirutin in ethanol, methanol, and acetone extracts. The hesperidin content showed a higher correlation (0.923-1.000) with all of the antioxidant activities than the narirutin content (0.741-0.958) except the reducing power, which means that the hesperidin in the extracts had a significant effect on antioxidant activities due to its high content and strong antioxidant activity. M'hiri et al. 48,49 also reported that hesperidin had higher antioxidant activity than narirutin due to the presence of a catechol group in the B-ring of the hesperidin molecule.

Conclusions
We optimized the extraction method for the highest recoveries of hesperidin and narirutin from ICUP, and the optimal extraction conditions were two sequential extractions at a temperature of 80.3 °C, ethanol concentration of 58.4% (v/v), and S/F ratio of 40 mL/g dry sample with a 30 min extraction time, where the hesperidin and narirutin yields were 92.1% and 97.2%, respectively. The ethanol extract showed higher antioxidant activities measured using nine different assay methods than methanol and acetone extracts. The ethanol extract showed very high scavenging activities against ROS. Hesperidin contents in ethanol, methanol, and acetone extracts showed a higher correlation with all of the antioxidant activities except the reducing power than narirutin contents. Therefore, the ethanol extract of immature C. unshiu pomace could be used as a flavonoid supplement for preventing human diseases caused by ROS. Further research is recommended to verify the anti-aging effect of those extracts based on experiments with cells and animals.