3.1. 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% H2
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
) of water was selected.
Typical HPLC-DAD chromatogram and UV spectrum of hesperidin standard are shown in Figure 1
a, as well as exemplary HPLC chromatogram of hesperidin quantification and separation from mandarin peels (Figure 1
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
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
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
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.
3.2. 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 (R2) and Lack of fit value. The obtained R2 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
, 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 3
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
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 using other solvents. Given the different varieties, as well as the geographical position and time of harvest, it is difficult to make an adequate comparison of our results.
Tumbas et al. [5
] extracted hesperidin from mandarin (Citrus reticulata
) peel with 70% (v
) aqueous solution of acetone during 2 h at a temperature of 40 °C at magnetic stirrer. The quantitative analysis showed that the obtained hesperidin content from mandarin peel was 31.42 mg/g of plant. In the case of kinnow peels, hesperidin was extracted with methanol and ethanol (50, 80, 100%) respectively. The obtained contents of hesperidin extracted with methanol were in the range from 44.38 ± 1.08 to 61.02 ± 1.17 µg/g of extract and with ethanol 75.66 ± 1.67 – 92.94 ± 1.23 µg/g of the extract [13