This paper reports a novel method for simultaneous extraction and in situ separation of flavonoids and alkaloids from mulberry leaves using a pH-responsive deep eutectic solvent (DES)/water system. Five novel DESs were synthesized, and isoquercitrin and deoxynojirimycin were respectively selected as the representative compounds of flavonoids and alkaloids to evaluate the extraction and separation efficiency. The optimal DES, namely [HexA (hexanoic acid)][MPD (2-methyl-2,4-pentanediol)], was identified, and the extraction and in situ separation conditions were optimized. After extraction and pH-induced phase separation, flavonoids were mainly enriched in the DES phase with alkaloids in the HO phase, and the distribution ratios were 6.69:1 and 1:9 ([HexA][MPD]:HO), respectively. Compared to traditional solvents, the [HexA][MPD]/HO system showed higher extraction efficiency of flavonoids and alkaloids with higher purity. Moreover, [HexA][MPD] possessed excellent reusability. This study demonstrates the application of pH-responsive DES/HO systems for simultaneous extraction and separation of natural products for the first time.
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