Natural Hydrophobic Deep Eutectic Solvent-Based Enhanced Extraction of Bioactive Compounds from Cannabis sativa L. Leaf for Pharmaceutical Applications

Cannabis sativa L. leaves (CSL) are a rich in bioactive compounds and known for their medicinal and recreational uses. In this study, a natural hydrophobic deep eutectic solvent (HDES) system composed of menthol and thymol (1:1) was employed for the efficient extraction of bioactive compounds from CSL. Extraction of bioactives was optimized at various conditions involving DES/ethanol ratio, temperature, and extraction time, as well as shaking speed through statistical models including response surface methodology (RSM) and artificial neural network (ANN). The maximum bioactive yield, equal to 70% (w/w) of powdered CSL, was achieved at optimized values of 5.5 mL DES, 4.5 mL ethanol, and 225 rpm shaking speed at 55 °C for 107.5 min. It was observed that slightly adjusting the shaking speed and temperatures customized the nature of bioactives with more antioxidant, antidiabetic, and antimicrobial properties. The extracts of CSL produced while applying natural HDES were found to be non-toxic during hemolytic assay. Overall, HDES when mixed with ethanol in 55:45 ratio produced CSL extracts with an ample level of phenolics (133.75 mg GAE/g) and flavonoids (120.05 mg QE/g). GC-MS analysis of CSL extracts produced by HDES revealed the presence of multiple bioactives like tetrahydrocannabivarin, cannabidiol, cannabinol, cannabidivarol, dl-menthol, levomenthol, and 4-hydroxy-3-methylacetophenone. Based on these findings, it can be concluded that HDES in combination with ethanol may work as an efficient extraction solvent to recover CSL bioactives without compromising their antioxidant features and safety for use in food and pharmaceutical applications.