Sustainable Green Procedure for Extraction of Hesperidin from Selected Croatian Mandarin Peels

: The peels of Citrus reticulata Blanco mandarin cultivars of di ﬀ erent Croatian varieties ( Zorica rana , Chahara , Okitsu , Kuno ) were extracted using 15 di ﬀ erent choline chloride-based deep eutectic solvents (DESs) at 50 ◦ C for 30 min and with 20% water addition. The extracts were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) to determine the most suitable DES for the extraction of hesperidin in the samples. The screening results indicated that choline chloride: acetamide (1:2) provided the most e ﬃ cient hesperidin extraction (112.14 mg / g of plant), while choline chloride:citric acid (1:1) solvent showed the lowest hesperidin yield (1.44 mg / g of plant). The Box–Behnken design was employed to optimize extraction parameters for each variety of mandarin peel, including extraction time, temperature and water content on hesperidin extraction. The results indicated that hesperidin content in mandarin peels was completely variety-dependent. Being a novel and e ﬃ cient green media for hesperidin extraction, deep eutectic solvents could also serve as promising solvent systems for the production of extracts rich in bioactive compounds.


Introduction
Citrus fruits are one of the most important crops with worldwide production, while citrus by-products represent a problem regarding their disposal due to the environmental risk.Traditionally, the waste was either burned, causing an increase in carbon dioxide and other greenhouse gasses, or used for cattle feed, but today more environmentally friendly approach was developed for obtaining the new high-value products.The citrus by-products include pulp, seeds and peels, where seeds present a valuable by-product, as well as the peels, due to their content of natural antioxidants, primarily flavonoids [1].The peels make the largest amount of total produced citrus by-products, and they can be utilized for different purposes due to their bioactive compounds content [2].
Composition of different constituents can vary regarding the diversity of citrus species and cultivars, as well as the genetic origin and the time of fruit collection.Therefore, citrus flavonoids can indicate the characteristic of each citrus species and variety [3].Compounds found in mandarin peels, such as flavanone glycosides and polymethoxy flavones are recognized as the major contributors to the biological activity of peels [4], with hesperidin being the most abundant flavonoid and the main functional compound [5].The studies have shown that it possesses hypoglycemic [6], antioxidant and cytotoxic effect against human cancer cell lines [7], anti-inflammatory [8] and antiproliferative activity [9].Hesperidin also exerts growth-inhibitory effects in different cancers, as well as significant antimetastatic activity [10], while possessing many other beneficial effects, which were reviewed by Garg et al. [11].
Since different processes can be applied for the extraction of bioactive compounds from citrus fruit, it is very important to find the most efficient extraction method to obtain the highest yield of selected bioactive compounds.However, the content of biologically active compounds can vary, considering the applied method and operating conditions.Some of the extraction methods require the use of toxic and ecologically unacceptable organic solvents.Some authors [12,13] investigated the extraction of hesperidin and other flavonoids from mandarin peels with isopropanol, ethanol and methanol, where ethanol is preferable for the application in the food processing due to the GRAS (generally recognized as dafe) characterization.Even though organic solvents can be applied for the extraction of specific compounds, their application in the food industry is unacceptable due to their toxicity and ecologically adverse effect.Therefore, more environmentally friendly solvents and methods should be applied.In the last few years, deep eutectic solvents (DESs) were proven a very efficient extraction media for obtaining different valuable compounds from various plant materials [14].They are characterized as green, nontoxic and cheap solvents formed of hydrogen bond acceptors (HBAs) in combination with hydrogen bond donors (HBDs).In eutectic mixtures, HBAs are quaternary ammonium or metal salts, while HBDs can be amides, carboxylic acids, alcohols or sugars.When mixed in a certain ratio and depending on the type of used HBD and HBA, DESs with desirable properties are produced [15].Recently, DESs are being used for the extraction of flavonoids from various fruits, vegetables and spices [16], as well as from Citrus aurantium L. [17] and the orange (Citrus sinensis) peels [18] where the DESs composition, concentration of added water, solid-to-liquid ratio and the effect of time, temperature and stirring speed on the extraction yield and composition of extracted flavonoids have been investigated.According to the results, DESs showed higher extraction yields and an increase in the solubility of active constituents compared to the other traditional solvents.The use of DESs as an extraction media has several advantages such as being environmentally friendly, with low cost and easy preparation of the solvents.
The work aimed to obtain the extracts rich in hesperidin from mandarin peels of Citrus reticulata Blanco cultivars of four selected Croatian variety including Zorica rana, Chahara, Okitsu and Kuno.Citrus reticulata Blanco is characterized by the greatest botanical variability [19].The extraction was performed with 15 different DESs where HBA was choline chloride, while HBDs were compounds such as urea, acetamide, butane-1,4-diol, glycerol, citric acid, malic acid, sorbitol, xylitol, oxalic acid, levulinic acid, ethylene glycol, malonic acid, thiourea, N-methyl urea and lactic acid.First, the screening for the most suitable DES in the extraction of the highest amount of hesperidin was performed.Hesperidin content was determined by high performance liquid chromatography with diode array detection (HPLC-DAD) analysis, and the best solvent was found to be choline chloride: acetamide DES.Then, the optimal conditions for the extraction of hesperidin were determined using response surface methodology (RSM).The influence of the extraction temperature (30, 50 and 70 • C), extraction time (30, 60 and 90 min) and the amount of added water (10, 20 and 30%) on the hesperidin content in the extracts obtained by choline chloride: acetamide DES was determined.This is the first report of the application of DESs for the extraction of hesperidin from four different Croatian variety of mandarin peels and the optimization of the process parameters.

Chemicals and Plant Material
The mandarin peels of Citrus reticulata Blanco cultivars of four different variety were obtained from small family farm Dalibor Ujević (Opuzen, Croatia) in 2017 during September (variety: Zorica rana), October (varieties: Chahara and Okitsu) and November (variety: Kuno).Before the extraction, the mandarin peels were dried and milled separately using a laboratory mill (IKA M 20 Universal mill).

Extraction of Hesperidin from Mandarin Peels with DESs
Dried and milled mandarin peels of each variety (Zorica rana, Chahara, Okitsu, Kuno) (50 mg) were mixed with 1 mL of the solvent, i.e., a mixture of DES with 20% (v/v) of demineralized water for screening.For initial screening, the mixture of DES and 20% of added water (v/v) was stirred at a temperature of 50 • C for 30 min.After optimization of the extraction process was performed, DESs were mixed with different amount of water (v/v) as emphasized in Table 2, at specified temperature and time.In both cases, prepared samples were stirred at 1500 rpm in aluminum block on a magnetic stirrer.After the extraction, the mixture was centrifuged at 6000 rpm for 5 min and then decanted.The supernatant (200 µL) was then diluted with 800 µL of methanol and filtered through the PTFE 0.45 µm filter before HPLC analysis.

HPLC Analysis of Hesperidin in the Extracts
Hesperidin was determined using a RP-HPLC method described in paper by Sun et al. [20] on a Agilent 1260 Infinity II (Analytical Instruments, CA, USA) with chromatographic separation obtained on a ZORBAX Eclipse Plus C18 (Agilent, CA, USA) column (100 × 4.6 mm, 5 µm) with isocratic elution of water as phase A and acetonitrile as phase B, at room temperature during 10 min.The flow rate was 1.0 mL/min, an injection volume of 20 µL was used, and UV detection wavelength was 210 nm.Hesperidin standard stock solutions were prepared in the methanol and calibration was obtained at seven concentrations (20.0-200.0mg/L).The linearity of the hesperidin calibration curve was confirmed by R 2 = 0.99955 with the limit of detection (LOD) of 0.001062 mg/L, quantification limit (LOQ) of 0.00354 mg/L and hesperidin retention time was 4.153 min.Results for obtained hesperidin content are given in Table 2.

Experimental Design
In order to evaluate the influence of three independent variables on hesperidin content in mandarin peel response, surface methodology technique (RSM) was applied.The process was analyzed and optimized with a Box-Behnken Design model in a quadratic function consisting of 17 randomized experimental runs with included five replicates at the central point.The effects of extraction time (30-90 min; X 1 ), temperature (30-70 • C; X 2 ) and water content (10-30 %; X 3 ) was investigated on the hesperidin yield (y) obtained by DES extraction.The coded and actual values of the independent variables used in experimental design are shown in Table 2. Statistical analysis and design of experiments were performed using Design-Expert ® software (ver.9, Stat-Ease Inc., Minneapolis, MN, USA) to determine the optimal extraction conditions for maximizing hesperidin content in the mandarin peel.

Development of the RSM Model
A second-order polynomial model is developed based on three input variables for hesperidin content in mandarin peel prediction and extraction process optimization: Time (X 1 ), temperature (X 2 ) and water content (X 3 ).Coded and actual levels of the independent process variables for RSM experimental design are shown in Table 2.A quadratic model for this study can be expressed by Equation ( 1): where y is the response or dependent variable, β 0 is the constant variable representing intercept, β i , β ii and β ij designate regression coefficients, X i and X j are inputs or independent variables.A relationship between individual factors X i is described with linear coefficients β i , cross product X ij with interaction coefficients β ij and quadratic variable X ii with quadratic coefficients β ii respectively.The RSM uses the least square method to estimate regression coefficients, which are used in model fitting.The adequacy of the fitted models is tested and evaluated through Lack of Fit, F-value and p-value of the ANOVA.

Screening of DES for Hesperidin Extraction
Since DESs are synthesized with various HBDs, they have a different physical, and chemical properties, like viscosity, pH, surface tension and polarity, and all of these parameters can have a significant influence on the extraction of hesperidin.Hence, in order to determine which DES is the most effective in the extraction of hesperidin, the extraction at constant process parameters was performed with 15 different DESs (Table 1).
The chosen extraction parameters were 50 • C, 20% H 2 O content and 30 min.The extraction time of 30 minutes and extraction temperature was chosen according to our own experience, as well as according to Liu et al. [17] who have shown that 30 min was the optimal time to extract hesperidin.Water content is important for reducing viscosity, but very high water content reduces the interaction between components, therefore, 20% (v/v) of water was selected.
Typical HPLC-DAD chromatogram and UV spectrum of hesperidin standard are shown in Figure 1a, as well as exemplary HPLC chromatogram of hesperidin quantification and separation from mandarin peels (Figure 1b).investigated on the hesperidin yield (y) obtained by DES extraction.The coded and actual values of the independent variables used in experimental design are shown in Table 2. Statistical analysis and design of experiments were performed using Design-Expert ® software (ver.9, Stat-Ease Inc., Minneapolis, MN, USA) to determine the optimal extraction conditions for maximizing hesperidin content in the mandarin peel.

Development of the RSM Model
A second-order polynomial model is developed based on three input variables for hesperidin content in mandarin peel prediction and extraction process optimization: Time (X1), temperature (X2) and water content (X3).Coded and actual levels of the independent process variables for RSM experimental design are shown in Table 2.A quadratic model for this study can be expressed by Equation (1): Where y is the response or dependent variable, β0 is the constant variable representing intercept, βi, βii and βij designate regression coefficients, Xi and Xj are inputs or independent variables.A relationship between individual factors Xi is described with linear coefficients βi, cross product Xij with interaction coefficients βij and quadratic variable Xii with quadratic coefficients βii respectively.The RSM uses the least square method to estimate regression coefficients, which are used in model fitting.The adequacy of the fitted models is tested and evaluated through Lack of Fit, F-value and p-value of the ANOVA.

Screening of DES for Hesperidin Extraction
Since DESs are synthesized with various HBDs, they have a different physical, and chemical properties, like viscosity, pH, surface tension and polarity, and all of these parameters can have a significant influence on the extraction of hesperidin.Hence, in order to determine which DES is the most effective in the extraction of hesperidin, the extraction at constant process parameters was performed with 15 different DESs (Table 1).
The chosen extraction parameters were 50 °C, 20% H2O content and 30 min.The extraction time of 30 minutes and extraction temperature was chosen according to our own experience, as well as according to Liu et al. [17] who have shown that 30 min was the optimal time to extract hesperidin.Water content is important for reducing viscosity, but very high water content reduces the interaction between components, therefore, 20% (v/v) of water was selected.
Typical HPLC-DAD chromatogram and UV spectrum of hesperidin standard are shown in Figure 1a, as well as exemplary HPLC chromatogram of hesperidin quantification and separation from mandarin peels (Figure 1b).As can be seen from Figure 2, there is a significant difference in the extraction ability among the used solvents at constant parameters, as well as between mandarin varieties.The highest amount  As can be seen from Figure 2, there is a significant difference in the extraction ability among the used solvents at constant parameters, as well as between mandarin varieties.The highest amount of hesperidin was extracted with ChCl-AA (102.0,68.3, 88.7, 112.1 mg/g of plant for Okitsu, Chahara, Kuno and Zorica rana respectively), while the lowest amount was extracted with ChCl-CiA (3.3, 1.4, 9.8, 4.2 mg/g of plant for Okitsu, Chahara, Kuno and Zorica rana), possibly due to increased viscosity of the solvent itself.Generally, the highest hesperidin content was extracted using basic DESs, such as ChCl-AA, ChCl-U and ChCl-NMeU.Similar results have been achieved with DESs such as ChCl-EG and ChCl-BDO, while between acid eutectic solvents most efficacious in the extraction of hesperidin were ChCl-LeA and ChCl-Mac, although basic DESs exhibit much higher yield (38.8-102.0,26.8-68.7,7.3-8.87,17.6-112.1 mg/g of plant for Okitsu, Chahara, Kuno and Zorica rana) than other DESs for the extraction of desired component (Figure 2).According to Budavari [21], hesperidin is soluble in dilute alkali and pyridine which is also proven in the paper by Al-Ashaal et al. [7], where extraction with alkaline solution gave the highest hesperidin yield.Given the higher extraction efficiency of solvent ChCl-AA compared to other basic solvents, the following was selected for further investigation and optimization.

Response Surface Analysis and Process Optimization
In order to optimize the extraction process, it is essential to evaluate the effects of several process variables (time, temperature and water content) and their interactions on the response (hesperidin content).Summarized results of the ANOVA are shown in Table 3 in order to evaluate the statistical significance of the proposed models for each investigated response.In this research, the investigated response is the extracted hesperidin content from the mandarin peel of different varieties.According to Budavari [21], hesperidin is soluble in dilute alkali and pyridine which is also proven in the paper by Al-Ashaal et al. [7], where extraction with alkaline solution gave the highest hesperidin yield.Given the higher extraction efficiency of solvent ChCl-AA compared to other basic solvents, the following was selected for further investigation and optimization.

Response Surface Analysis and Process Optimization
In order to optimize the extraction process, it is essential to evaluate the effects of several process variables (time, temperature and water content) and their interactions on the response (hesperidin content).Summarized results of the ANOVA are shown in Table 3 in order to evaluate the statistical significance of the proposed models for each investigated response.In this research, the investigated response is the extracted hesperidin content from the mandarin peel of different varieties.Based on the obtained results, the regression models for all investigated responses of Citrus reticulata varieties were significant (p-value < 0.05), while the quality of the models developed was evaluated based on the coefficients of determination (R 2 ) and Lack of fit value.The obtained R 2 values for all models developed was in the range from 0.8429 to 0.9042 with non-significant Lack of fit indicated adequate representation between input parameters and observed variable, in this case, hesperidin content in mandarin peel of different varieties.The model developed for the variety Okitsu implies no significant influence of the extraction time or water content on the process of hesperidin extraction using DESs.
However, temperature and quadratic terms of temperature and water content variables showed significant influence on the extraction process as given in Table 4.In addition, the temperature was a significant parameter for all models except for the model developed for Citrus reticulata variety Chahara, showing increased hesperidin yield with the increase of the temperature.An interesting observation has been made about the influence of the water content for varieties of Chahara and Zorica rana where water content parameter showed a significant effect on the hesperidin yield from mandarin peels (Figure 3b,c).
As can be seen, the increase in water content causes the increase in hesperidin yield until it reaches its maximum (mostly around 20%).After that, the additional increase in water content (above 20%) causes the hesperidin yield to decrease.This phenomenon could be potentially explained by the fact that higher water content weakens the interactions between DESs and hesperidin.
Water content is important, since it reduces the viscosity of the solvent, thereby improving the mass transfer and extraction process.However, the excessive water content can reduce the interaction between the solvent components, as well as the solvent and the desired component interactions [22].Addition of water up to 20% reduces the viscosity of the solvent contributing to the better extraction, and with further increase in the water content above 20% the needed interactions are reduced, as well as the extracted content of hesperidin.Since hesperidin is a component that is very poorly soluble in water, it is understandable that the increase in water addition decreases its solubility in the solvent [23].One of the main goals of this study is to optimize DES extraction processes by maximizing hesperidin yield using desirability approach.
For the variety Okitsu, the optimal conditions for hesperidin extraction were estimated to be at time 90 min, at a temperature of 68.14 • C, and water content of 13.83%.Moreover, optimal conditions were calculated to be at 45.40 min, 69.70 • C and water content of 10.67% for variety of Chahara, while 88.79 and 54.72 min, 55.02 and 69.66 • C, 19.73 and 14.86% were calculated as optimal conditions for hesperidin extraction by DES of choline chloride and acetamide in 1:2 molar ratio for Kuno and Zorica rana, respectively.Predicted data obtained with RSM analysis for each investigated variety were experimentally verified with a good agreement to the experimental values within a deviation of ± 5%.Moreover, in order to evaluate the obtained models, a graphical comparison was made of the actual versus predicted values for responses of the four different mandarin varieties, as shown in Figure 4.
In addition, the temperature was a significant parameter for all models except for the model developed for Citrus reticulata variety Chahara, showing increased hesperidin yield with the increase of the temperature.An interesting observation has been made about the influence of the water content for varieties of Chahara and Zorica rana where water content parameter showed a significant effect on the hesperidin yield from mandarin peels (Figure 3b,c).As can be seen, the increase in water content causes the increase in hesperidin yield until it reaches its maximum (mostly around 20%).After that, the additional increase in water content (above 20%) causes the hesperidin yield to decrease.This phenomenon could be potentially explained by the fact that higher water content weakens the interactions between DESs and hesperidin.
Water content is important, since it reduces the viscosity of the solvent, thereby improving the mass transfer and extraction process.However, the excessive water content can reduce the interaction between the solvent components, as well as the solvent and the desired component interactions [22].Addition of water up to 20% reduces the viscosity of the solvent contributing to the better extraction, and with further increase in the water content above 20% the needed interactions are reduced, as well as the extracted content of hesperidin.Since hesperidin is a component that is very poorly soluble in water, it is understandable that the increase in water addition decreases its solubility in the solvent [23].One of the main goals of this study is to optimize DES extraction processes by maximizing hesperidin yield using desirability approach.
For the variety Okitsu, the optimal conditions for hesperidin extraction were estimated to be at time 90 min, at a temperature of 68.14 °C, and water content of 13.83%.Moreover, optimal conditions were calculated to be at 45.40 min, 69.70 °C and water content of 10.67% for variety of Chahara, while 88.79 and 54.72 min, 55.02 and 69.66 °C, 19.73 and 14.86% were calculated as optimal conditions for hesperidin extraction by DES of choline chloride and acetamide in 1:2 molar ratio for Kuno and Zorica rana, respectively.Predicted data obtained with RSM analysis for each investigated variety were experimentally verified with a good agreement to the experimental values within a deviation of ± 5%.Moreover, in order to evaluate the obtained models, a graphical comparison was made of the actual versus predicted values for responses of the four different mandarin varieties, as shown in Figure 4.

Figure 3 .
Figure 3. Response surface plots showing the effects of temperature, extraction time and water content on the extraction yield of hesperidin in different mandarin varieties: (a) Okitsu; (b) Chahara; (c) Kuno; (d) Zorica Rana.

Figure 3 .
Figure 3. Response surface plots showing the effects of temperature, extraction time and water content on the extraction yield of hesperidin in different mandarin varieties: (a) Okitsu; (b) Chahara; (c) Kuno; (d) Zorica Rana.

Figure 4 .
Figure 4. Graphical comparison of actual and predicted values of the extracted hesperidin content for each variety of mandarin: (a) Okitsu; (b) Chahara; (c) Kuno; (d) Zorica Rana.A literature search did not reveale data on DESs extraction and optimization of the parameters for hesperidin from Citrus reticulata, but there few papers that investigate the possibility of extraction

Table 1 .
List of DESs prepared and tested for the extraction of hesperidin from mandarin peels.

Table 2 .
Coded and actual levels of the independent variable for the Box-Behnken design with experimental hesperidin yields.

Table 3 .
Analysis of variance (ANOVA) of second-order polynomial models for hesperidin content in the mandarin peels.** p < 0.01 highly significant; * 0.01 ≤ p < 0.05 significant; p ≥ 0.05 not significant. a

Table 4 .
Estimated coefficients of the regression model for hesperidin content.