Development and optimization of different extraction and analytical methods on Humulus lupulus L. with molecular networks, quantifications and desirability approaches

Abstract

Specific natural compounds of Humulus lupulus L. (hop cones) have significant applications both in food and pharmaceutical industries. The effectiveness of these applications depends on the extraction methods used, which can vary in the yield and metabolite profile of the extract. This study focuses on the development of green solvent extraction methods in order to adapt the process to industrial-scale extraction in other plant species. Four extraction techniques were employed, including maceration (simple maceration (SM) and triphasic-SE), ultrasound (UAE), high pressure (PLE), microwaves (MAE). Two of them were also combined (UAE+SM). Ethanol and a combination of ethanol and water were mostly used. Triphasic extraction was performed with classical solvents. The results were compared in term of yield, total assays (polyphenols, flavonoids, and chlorophylls), metabolite profile, LC-MS/MS quantification, and molecular networking. UAE led to high yield with reduced extraction time compared to maceration, and this result was further amplified with pressure-assisted extraction (PLE) with a 3- and 6-fold increase in yield and time reduction, respectively. The yields were correlated with higher quantities of molecules of interest, such as α/β acids, polyphenols, and flavonoids. For instance, the MAE and PLE techniques provided high content of flavonoids and polyphenols (2 and 3-fold), xanthohumol (6 and 4-fold) compared to SE. The use of alternative extraction methods alone or coupled significantly improved the yields and contents. LC-MS/MS allowed for the monitoring of various metabolite concentrations, which were also found in molecular networks, and that facilitating the selection of the most appropriate technique. A descriptive statistical analysis was used to provide a global vision between the different extractive methods and determine the best experimental conditions. Among the various treatments tested, the global desirabilities from 65.5 % and 77.4 % were achieved with PLE followed by UAE-B+SM (64.5 %). They emerged as the most effective approach providing a balance between mass yields, content and concentration of metabolites of interest.