## 3 Results and Discussion
### Selection of DESs for Mixture Design Studies Based on the ISO Extraction Yields  
The ISO content of CC, CJ, and GB were determined to be 861, 276, and 118 ug/g, respectively, when using 70% w/w DES 1. Because CC had the highest ISO level among the three trees, it was therefore used as a model for comparing the extraction efficiency of the prepared DESs. The ISO levels post extraction with DESs 1-11 (746-915 ug/g) were significantly (\(p<0.05\)) higher than those with hot water (705 ug/g) (Figure 1). However, they were significantly (\(p<0.05\)) lower than those acquired using 70% methanol (1086 ug/g). Glycerol generally served as a good component for DESs that are effective for ISO extraction, whether its counterpart was a sugar or an organic acid. Moreover, organic acids appeared to be superior components to glycerol, and DESs 8-10 containing malic acid or lactic acid resulted in the highest ISO extraction yields. However, DESs with organic acids are usually highly viscous and thus difficult to handle [(24)], and so were the malic acid-based DESs (DESs 8 and 9). DES 10, with yield similar to those of DESs 8 and 9, was excluded from further consideration because of the low lactic acid purity (\(\sim\)90%). Accordingly, DES 1 (ISO yield, 861 ug/g), which could be synthesized from glycerol and xylitol and handled with ease, was deemed the reasonable extraction solvent for the tree leaves.  
Figure 1: Comparison of isoquercetin (ISO) levels post extraction with DESs, to those post extraction with 70% methanol and water. The water content of all the tested DESs was 30% w/w. Error bars indicate standard error of the mean (n = 3).  
Before applying DES 1 to the mixture design studies, the effects of several factors on extraction efficacy were examined. First, in varying the molar ratio of glycerol to xylitol from 1:1 to 5:1, at 30% water, we found that DES 1 with a 5:1 ratio (designated as DES 1-5) had the highest efficiency, although it was not significantly different from that of DES 1 with a 4:1 ratio (DES 1-4) (Table S1). Then, the effect of the water content of DES 1 was assessed by measuring ISO yields with DES 1 with various water contents (10-50% w/w). We found that 30% water exhibited the highest yield (946 \(\mathrm{\SIUnitSymbolMicro g}\)/g), while 50 and 10% water resulted in \(<\)900 \(\mathrm{\SIUnitSymbolMicro g}\)/g and \(\sim\)700 \(\mathrm{\SIUnitSymbolMicro g}\)/g, respectively (Table S2). Based on the faster synthesis time and lower viscosity, DES 1-5 was regarded as desirable, and its 70% aqueous solution was used as the final extraction solvent in the mixture design studies. It is apparent that the selected solvent (70% w/w DES 1-5) is less economical than water as an extraction medium. However, it is noteworthy that the extraction efficiency of the selected solvent was achieved without any heat treatment, and that the individual DES components can act as functional ingredients in cosmetics [20]. The water extraction, which had to be conducted at a boiling temperature for improved efficiency, might cause degradation of unstable compounds during extraction. These factors could provide merit for the use of the DES solution in this study.