Extraction of Gnetum gnemon L. (Melinjo) Leaves Using Deep Eutectic and Conventional Solvents: Phytochemical Contents, Antioxidant Capacity, and Toxicity Assessment

Abstract

Gnetum gnemon L. (Melinjo) is widely recognised for its rich bioactive compounds with potential health benefits. This study evaluates the effectiveness of deep eutectic solvents (DESs) and conventional solvents using ultrasound-assisted extraction on the extraction efficiency, antioxidant activity, and toxicity of G. gnemon leaf extracts (GGLE). Four DESs, namely choline chloride-lactic acid (ChCl-LA), choline chloride-glycerol (ChCl-Gly), betaine-lactic acid (Bet-LA), and L-proline-citric acid (LP-CA), were compared with conventional solvents, namely water, methanol, and ethanol. Extraction performance was evaluated based on crude extract yield (CEY), phytochemical content through chlorogenic acid content (CAC), total phenolic (TP), and total flavonoid (TF), as well as antioxidant activity assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. Toxicity was assessed using the brine shrimp lethality assay (BSLA). Among all solvents, ChCl-LA yielded the highest CEY (20.23 ± 0.68%), CAC (13.96 ± 0.31 µg/g), TP (92.54 ± 0.74 mg GAE/g), DPPH (84.95 ± 1.49%), ABTS (91.25 ± 0.34%), and FRAP (481.14 ± 2.31 mg TE/g) (p < 0.05), while exhibiting no toxicity with a high LC₅₀ value (6,339.43 µg/mL). Nonetheless, ethanol yielded the highest TF (2.98 ± 0.47 mg QE/g), followed by ChCl-Gly (2.45 ± 0.27 mg QE/g), although non-significant (p > 0.05), with ChCl-LA (2.14 ± 0.27 mg QE/g) placed as the third highest. Overall, DESs, particularly ChCl-LA, demonstrated promising extraction efficiency and non-toxicity compared to conventional solvents, supporting their potential as greener alternatives for extracting bioactive compounds from G. gnemon leaves. HIGHLIGHTS Deep eutectic solvents (DESs) demonstrated greater effectiveness than conventional solvents in extracting bioactive compounds from Gnetum gnemon The combination of choline chloride and lactic acid (ChCl-LA) produced the highest crude extract yield, exhibited strong antioxidant activity, and showed no observable toxicity. Extracts obtained using DESs and water were non-toxic, indicating their safety for potential application in functional foods and nutraceutical products. GRAPHICAL ABSTRACT