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 pre-vention 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 degrees 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 chromatog-raphy (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.