Natural Deep Eutectic Solvents (NADESs) for the Extraction of Bioactive Compounds from Quinoa (Chenopodium quinoa Willd.) Leaves: A Semi-Quantitative Analysis Using High Performance Thin-Layer Chromatography

Abstract

Natural deep eutectic solvents (NADESs) have emerged as a promising eco-friendly alternative to petrochemicals for extracting plant metabolites. Considering that the demand for sustainable "green" ingredients for industrial applications is growing, those solvents are purported to develop extracts with interesting phytochemical fingerprints and biological activities. Given the interest in flavonoids from Chenopodium quinoa Willd. leaves, an efficient "green" extraction method was developed by investigating eight NADESs with defined molar ratios, i.e., malic acid-choline chloride (chcl)-water (w) (1:1:2, N1), chcl-glucose-w (5:2:5, N2), proline-malic acid-w (1:1:3, N3), glucose-fructose-sucrose-w (1:1:1:11, N4), 1,2-propanediol-chcl-w (1:1:1, N5), lactic acid-glucose-w (5:1:3, N6), glycerol-chcl-w (2:1:1, N7), and xylitol-chcl-w (1:2:3, N8). Rheological measurements of all NADESs confirmed their pseudoplastic behaviors. To improve the extraction processes, differential scanning calorimetry (DSC) allowed us to determine the maximum amount of water that could be added to the most stable NADES (N1, N2, N3, and N4; 17.5%, 20%, 10%, and 10% w/w, respectively) to lower their viscosities without disturbing their eutectic environments. The phytochemical compositions of NADES extracts were analyzed using high-performance thin-layer chromatography (HPTLC), and their free radical scavenging and α-amylase inhibitory properties were assessed using HPTLC-bioautography. N2, diluted with 20% of water, and N7 presented the best potential for replacing methanol for an eco-friendly extraction of flavonoids, radical scavengers, and α-amylase inhibitors from quinoa leaves. Their biological properties, combined with a good understanding of both thermal behavior and viscosity, make the obtained quinoa leaf NADES extracts good candidates for direct incorporation in nutraceutical formulations.

Keywords: Amaranthaceae; antioxidant activity; eco-extraction; high-performance thin layer chromatography-bioautography; natural deep eutectic solvents.

Figure 1

DSC Thermogram obtained at 10 °C/min for eight undiluted NADESs. Ramps: (I) heating from 30 °C to 80 °C; (II) cooling from 80 °C to −80 °C; and (III) heating from −80 °C to 30 °C. N5 exhibits exo and endo transitions due to crystallization and melting in the reheating scan.

Figure 2

(A) Effect of water dilutions on the thermal stability of NADES N2. (B) Effect of water dilutions on the thermal stability of NADES N3. The solid circle indicates how increasing the water content in the dilution of the eutectic mixture leads to more pronounced transitions in the thermogram.

Figure 3

(A) Effect of water dilutions on the thermal stability of NADES N1. (B) Effect of water dilutions on the thermal stability of NADESs N4. The curves for 50% (w/w) of water are not included in order to observe the smaller transitions. The solid circle indicates how increasing the water content in the dilution of the eutectic mixture leads to more pronounced transitions in the thermogram.

Figure 4

Log plots of viscosities as a function of shear rate for the studied NADESs (40 °C). N4 is not included because it crystallized before measurements.

Figure 5

Log plots of viscosities as function of shear rate for N2 (choline chloride:glucose:water, molar ratio 5:2:5) diluted with different amounts of water (40 °C).

Figure 6

Colors and HPTLC flavonoid fingerprints of the eight NADES extracts (from N1E to N4E, NADES diluted with water as per Table 1; from N5E to N8E, undiluted NADES) obtained from quinoa leaves (sample/solvent ratio, 1:20 w/w; application volumes, 4 µL). Mobile phase: formic acid–water–methyl ethyl ketone–ethyl acetate (10:10:30:50, v/v/v/v). Derivatization with NP and PEG; examination under UV365 nm. Tracks: (1) aqueous extract; (2) methanolic extract; (3) methanol:water (80:20, w/w) extract; from (4) to (11) SPE recovered NADES extracts, from N1R to N8R.

Figure 7

Peak profiles generated from Figure 6, i.e., the HPTLC plate examined under UV365 nm after derivatization with NP and PEG. Tracks: (1) aqueous extract; (3) methanol:water (80:20, w/w) extract; (5), (8), (10), and (11) SPE recovered NADES extracts N2R, N5R, N7R, and N8R.

Figure 8

Free radical scavenging activity of the 8 NADES extracts (from N1E to N4E, NADESs diluted with water as per Table 1; from N5E to N8E, undiluted NADESs) obtained from quinoa leaves (sample/solvent ratio, 1:20 w/w; application volumes, 6 µL). Mobile phase: formic acid–water–methyl ethyl ketone–ethyl acetate (10:10:30:50, v/v/v/v). Derivatization with DPPH● for 90 s (A) and 120 min (B) and examination under visible light. Tracks: (1) aqueous extract; (2) methanolic extract; (3) methanol:water (80:20, w/w) extract; from (4) to (11) SPE recovered NADES extracts, from N1R to N8R. Low-intensity bands marked with a black dotted line.

Figure 9

α-amylase inhibitory activity of leaf quinoa extracts (sample/solvent ratio, 1:20 w/v; application volumes, 8 µL). Mobile phase: formic acid–water–methyl ethyl ketone–ethyl acetate (10:10:30:50, v/v/v/v). Derivatization with α-amylase, starch, and iodine; examination under visible light. Tracks: (1) methanolic extract; from (2) to (5) SPE recovered NADES extracts, from N1R to N4R. The red marks indicate α-amylase inhibition zones. The large blue smear at the bottom of the plate (Rf from 0.0 to 0.2) is due to inhibition of α-amylase by the formic acid of the mobile phase.