Use of [C4mim]Cl for efficient extraction of caffeoylquinic acids from sweet potato leaves

Sweet potato, Ipomoea batatas, is a widely cultivated vegetable worldwide. The leaves contain polyphenolic natural products called caffeoylquinic acids (CQAs), which possess biological activities including inhibition of aggregation of amyloid peptides. The present study describes an efficient extraction and isolation procedure for CQAs from sweet potato leaves using a cellulose-dissolving ionic liquid. The results showed that, compared to methanol, use of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) allowed the extraction of a 6.5-fold greater amount of CQAs. This protocol will enable the efficient extraction of other organic compounds and biopolymers from natural materials.

Next, the extraction procedure using the cellulose-dissolving ionic liquid [C 4 mim]Cl was carried out as follows. The crushed sweet potato leaves were added to extraction solvents, and the solution was stirred for 1 hour: [C 4 mim]Cl at 150 °C, [C 4 mim]Cl/MeOH (1:1 w/w, 3:1 w/w) at 100 °C, [C 4 mim]Cl/H 2 O (1:1 w/w, 3:1 w/w) at 120 °C, and [C 4 mim]Cl/MeOH/H 2 O (2:1:1 w/w/w) at 100 °C. Then, MeOH was added to the solution, and the mixture was stirred at room temperature for 30 seconds. The solution was filtered, evaporated, and the extract was dissolved in H 2 O/MeOH (7:3 v/v). The solution was washed with n-hexane three times. Removal of organic solvents afforded the crude extract as a [C 4

mim]Cl solution.
Quantitative analysis of the obtained extracts was performed using reversed-phase high performance liquid chromatography (RP-HPLC). For the analysis, the quantity of CQAs in individual extracts was calculated using a calibration curve. To prepare the calibration curves, 3,4-, 3,5-, and 4,5-diCQAs were purchased from a commercial supplier, whereas 3,4,5-triCQA was prepared by organic synthesis according to literature procedure starting from quinic acid 17  mim]Cl afforded the best yield (0.78%), while yields for 3,4,5-triCQA using [C 4 mim]Cl/MeOH (1:1 w/w or 3:1 w/w) and [C 4 mim]Cl/H 2 O (1:1 w/w) were 0.26% and 0.30%, respectively, 2.9 and 1.9 times greater than those achieved using MeOH (0.09%) and H 2 O (0.16%). As the best comparison among the obtained results, the extraction yield of 4,5-diCQA using [C 4 mim]Cl (0.78%) was 6.5 times better than that using MeOH (0.12%).  Factors affecting the extraction results include the solubility of the cellulose making up the plant cell wall, the solubility of associated compounds in the extraction solvent, and the efficiency of stirring process. Because MeOH and H 2 O are poor solvents for cellulose, cellulose solubility decreases in a mixed solvent such as [C 4 mim] Cl/MeOH or [C 4 mim]Cl/H 2 O. However, this tendency did not affect overall extraction yields. The extraction yield for 3,5-diCQA using MeOH was much better than that using H 2 O, while the yields for other CQAs using H 2 O were much better than those using MeOH. This difference was probably due to the formation of micelle-like aggregation by CQAs except for 3,5-diCQA in H 2 O derived from the orientation of caffeoyl groups. The relatively long distance between the two caffeoyl groups in 3,5-diCQA may prevent the aggregation.
To investigate the stirring efficiency, the viscosity of the extraction solvents was measured (Fig. 4). The ionic liquid [C 4 mim]Cl possessed extremely high viscosity as expected (Fig. 4a). A comparison between [C 4 mim] Cl/MeOH (1:1 w/w) and [C 4 mim]Cl/MeOH (3:1 w/w) indicated that greater amounts of [C 4 mim]Cl promoted greater viscosity (Fig. 4b), and a comparison between [C 4 mim]/H 2 O (1:1 w/w) and [C 4 mim]/H 2 O (3:1 w/w) also indicated the same tendency (Fig. 4c) Furthermore, isolation of CQAs from the [C 4 mim]Cl layer was investigated using a salting-out and decantation technique. The [C 4 mim]Cl extract was added to a saturated sodium chloride solution, followed by addition of an extractipm solvent such as EtOAc/MeOH (10:1 v/v), EtOAc/MeOH (5:1 v/v), EtOAc/MeOH/HCO 2 H (100:10:1 v/v/v), or EtOAc/MeOH/HCO 2 H (100:20:1 v/v/v). The organic layer was collected from the mixture by decantation. This procedure was repeated four times, and the combined organic layers were removed to obtain the raw CQAs. The CQAs were subjected to HPLC analysis, and the amounts of CQAs obtained were compared to the original amounts of CQAs in [C 4 mim]Cl layer ( Table 1).
Extraction of CQAs from the [C 4 mim]Cl layer was attempted using an EtOAc/MeOH (10:1 v/v) extraction solvent (Table 1, entry 1), assuming a salting-out effect by saturated sodium chloride. However, the method afforded low yields with slight contamination of the [C 4 mim]Cl. An increase in MeOH promoted the solubility of the   CQA extraction yields (entry 4). Because small amount of [C 4 mim]Cl was found in the resultant CQAs under the condition of entry 4, the condition of entry 3 would be the best for the extraction of CQAs from [C 4 mim]Cl layer. Thus, dissociation of a proton from formic acid probably prevented dissociation of protons from the CQAs, increasing the solubility in organic solvents (EtOAc).

Conclusions
In summary, a method for the efficient extraction and isolation of CQAs from sweet potato leaves was developed using the cellulose-dissolving ionic liquid [C 4 mim]Cl. The use of [C 4 mim]Cl allowed the CQAs to be extracted more efficiently than using conventional MeOH or H 2 O solvents. The results demonstrated that the extraction yield of 4,5-diCQA using [C 4 mim]Cl (0.78%) was 6.5 times better than that using MeOH (0.12%). This method is expected to be applicable to the extraction and isolation of other organic compounds and biopolymers from natural materials. Sweet potato leaves (Sui-oh) were purchased from Shijoukai, Midorien (Fukuoka, Japan) 16 . The HPLC analyses were performed using a JASCO instrument equipped with a multiwavelength detector (MD-2010), semi-micro HPLC pump (PU-2085), autosampler (AS-2057), and column thermostat (CO-2060). Optical rotation was measured using a JASCO P-2200 digital polarimeter at the sodium D line (λ = 589 nm) and is reported as follows:
Extraction of CQAs from sweet potato leaves. The CQAs were extracted from sweet potato leaves using MeOH or H 2 O as a reference. The sweet potato leaves (1 g) were treated with liquid nitrogen and then crushed into approximately 0.5 × 0.5 cm 2 pieces in a mortar. Then, the crushed leaves were added to MeOH (16 g) or H 2 O (16 g), and the solution was stirred under reflux at around 80 °C or 120 °C, respectively, for 1 hour. The resulting solution was filtered with Celite 545 to remove the leaf residue. The filtrate was evaporated, and the extract was dissolved in H 2 O/MeOH (7:3 v/v). The solution was washed with n-hexane three times to remove undesired hydrophobic materials. Removal of the organic solvent afforded a crude extract as a brownish solid. Analyses of the extracts obtained were performed using RP-HPLC.
Extraction using the cellulose-dissolvable ionic liquid [C 4 mim]Cl was carried out as follows. Crushed sweet potato leaves (1 g) were added to the extraction solvents ( The solution was filtered with Celite 545, the filtrate was evaporated using a rotary evaporator, and then the extract was dissolved in H 2 O/MeOH (7:3 v/v). The solution was washed with n-hexane three times. After evaporation, the crude extract derived from an ionic liquid method was obtained as a [C 4 mim]Cl solution. Analyses of the extracts obtained were performed on RP-HPLC.
HPLC analysis of CQAs. Quantitative analyses of the extracts obtained were performed using an RP-HPLC system [column: YMC-Pack ODS-AM (150 × 4.6 mm); mobile phase: 0.2% formic acid aqueous solution/acetonitrile (gradient), flow rate: 1.0 mL/min; detection: 326 nm; injection amount: 10 μL; temperature: 40 °C] 3 . The quantity of CQA from individual extraction experiments was calculated using a calibration curve. The equations 1-4 for each CQA were obtained using the formulas below, where x represents peak area and y represents the amount of CQA (mg):  The extraction yields were calculated as the [quantity of CQAs] per [original quantity of leaves].