*2.1. Chemical Profiling of PUL Extracts*

A total of 30.02 g of dried PUL was extracted to obtain 4.24 g of freeze-dried extract powder (yield: 14.12%). To analyze the active compounds present in the PUL, the extract was subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. On performing LC-MS/MS analysis, valid peaks corresponding to the flavonoid components were detected in the range of 35–37 min at a wavelength of 270 nm. As can be observed in Figure S1A, the *m*/*z* value of [M-H]- was 609.15 at 35.0 min. The *m*/*z* value of [M-H]− was 463.09 at 35.2 min and 35.6 min (Figure S1B,C). From the results of the MS analysis, rutin, hyperoside, and isoquercitrin were determined to be the flavonoid components of PUL. In this study, the HPLC-MS/MS results show that rutin, hyperoside, and isoquercitrin are the active flavonoid components present in the PUL extracts. To confirm the presence of the flavonoids in the PUL extracts, standard samples of rutin, hyperoside, and isoquercitrin were subjected to HPLC analysis equipped with a Waters 2996 PDA detector, and the resulting chromatograms were compared with that obtained for the PUL extract (Figure 1). The retention times of the standard rutin, hyperoside, and isoquercitrin samples are consistent with those of the peaks 1, 2, and 3 of the PUL extract.

**Figure 1.** HPLC chromatograms of PUL extract and standard samples. Detection interval of (**A**) extract, (**B**) rutin, (**C**) hyperoside, and (**D**) isoquercitrin.

#### *2.2. Preparation of DES and Selection of DES by Extraction Efficiency*

Five types of DESs were synthesized, using CC as the HBA. Different dicarboxylic acids were employed as the HBDs. All the HBDs contain two carboxyl groups, but differ in the total number of carbon atoms. The efficiencies of the solvent extractions of the flavonoids from PUL, using the various DESs, are listed in Table 1. The extraction efficiency of the flavonoids is expressed as the sum of the extraction contents of three components: rutin, hyperoside, and isoquercitrin. Three standard samples of each extract were evaluated using HPLC with a correlation coefficient of 0.99. The contents of the flavonoids in the extract were determined using this standard curve. All the extraction efficiencies with the DESs were higher than those obtained with water (used for comparison), except for that pertaining to the DES synthesized using adipic acid. The extraction efficiencies of PUL with the malonic- and glutaric acid (GA)-based DESs were significantly higher than those with the other DESs. Therefore, the DES prepared using GA and CC was selected for the extraction of PUL prior to the optimization of the extraction condition. The HBDs and HBAs in the DES can interact with the cellulose and lignin in the plant cell walls to promote loosening of the cell wall structure and enable easy extraction of the intracellular components. A higher maximum extraction efficiency was achieved with the DES than without it, which was consistent with previous reports [27]. In addition, the current results show that when CC is used as the HBA, the extraction efficiency varies with the types of HBD used. Higher extraction efficiencies were achieved using DESs based on structurally similar dicarboxylic acids with odd numbers of carbon atoms. It has been confirmed that the structures of the HBDs affect the extraction efficiency, and it can be observed in Table 1 that the molar ratios vary depending on the structures. The current results also indicate that the eutectic temperature is determined by the structure of the HBD. Field emission scanning electron microscopy (FE-SEM) studies indicated the penetration of trace amounts of the solvents on the PUL surface during the extraction process. The FE-SEM images of the pre- and post-extraction PUL samples are shown in Figure 2. As can be observed in Figure 2A, there was no solvent penetration on the surface of the pre-extraction PUL, while traces of solvent penetration were observed when the extraction was performed with water (Figure 2B). The FE-SEM images of the PUL surfaces after DES extraction (Figure 2C,D) indicate the occurrence of sufficient solvent penetration. The high solubility of the plant cell wall components, such as lignin, cellulose, and flavonoids, in the DES facilitated the penetration of the solvent, which led to structural changes in the overall leaf surface. The efficient penetration of plant tissues by the DES prepared using GA and CC proves that it can serve as an environmentally friendly solvent for isolating natural active components. Based on the penetration, it can be concluded that the synthesized covalent solvent is sufficiently functional.


**Table 1.** Extraction efficiency of flavonoids from *PUL*.

\* Flavonoid contents are the sum of contents of the three components (rutin, hyperoside, and isoquercitrin).

**Figure 2.** Configuration of PUL surface after extraction using different solvents. (**A**) PUL surface before extraction; (**B**) PUL surface after extraction with water; (**C**) PUL surface after extraction with DES synthesized using CC and malonic acid; (**D**) PUL surface after extraction with DES synthesized using CC and GA.
