Liquid-liquid chromatography separation of hemp terpenes with repellent properties against Hyalomma marginatum: A multi-methodological approach

Tick-borne diseases raise serious health concerns for both humans and animals. Tick control and disease prevention largely depend on synthetic acaricides and repellents, often incriminated for high toxicity. To find alternative, safe, and efficient agents against ticks, research on plant-based volatile terpenes constitutes a promising direction. This study aimed to separate and identify hemp flower compounds with repellent properties against Hyalomma marginatum (Acari: Ixodidae). Produced by supercritical CO₂ extraction under different pressure (100–110 bar) and temperature (40–60 °C) conditions, the hemp extracts were tested in a moving object bioassay. The extract with the highest repellent power was further subjected to batch liquid-liquid chromatography (LLC). The biphasic solvent system (n-heptane/methanol/acetonitrile 10/5/10, v/v/v) for separating the major hemp terpenes was selected by combining the COSMO-RS-based distribution coefficient screening with experimental validation. Bisabolol (>99%), caryophyllene oxide (>98%), and caryophyllene/humulene (3.5/1, w/w, >95% purity) were thus isolated and assessed in the H. marginatum bioassay. The activity of bisabolol (20% w/w in hexane) was comparable to the synthetic repellent DEET at the same concentration. For the large-scale purification of bisabolol, trapping multiple dual mode, a semi-continuous flow-reversal LLC operating mode, was proposed and evaluated. Bisabolol was obtained with a solvent consumption 2.6-times lower and productivity 2.7-times higher than the batch process. In conclusion, using a multi-methodological computer-aided approach, bisabolol was efficiently identified and isolated as a good tick repellent agent from hemp flowers.