Histo-cytochemistry and scanning electron microscopy for studying spatial and temporal extraction of metabolites induced by ultrasound. Towards chain detexturation mechanism.

There are more than 1300 articles in scientific literature dealing with positive impacts of Ultrasound-Assisted Extraction (UAE) such as reduction of extraction time, diminution of solvent and energy used, enhancement in yield and even selectivity, intensification of diffusion, and eliminating wastes. This present study aims to understand what are the mechanism(s) behind these positive impacts which will help to design a decision tool for UAE of natural products. Different microscopic observations (Scanning Electron Microscopy (SEM), Environmental Scanning Electron Microscopy (e-SEM), Cyto-histochemistry) have been used for spacial and temporal localization of metabolites in rosemary leaves, which is one of the most studied and most important plant for its antioxidant metabolites used in food industry, during conventional and ultrasound extraction. The study permits to highlight that ultrasound impacted rosemary leaves not by a single or different mechanisms in function of ultrasound power, as described by previous studies, but by a chain detexturation mechanism in a special order: local erosion, shear forces, sonoporation, fragmentation, capillary effect, and detexturation. These detexturation impacts followed a special order during ultrasound treatment leading at the end to the total detexturation of rosemary leaves. These mechanisms and detexturation impacts were identified in glandular trichomes, non-glandular-trichomes and the layer adaxial and abaxial cuticle. Modelling metabolites diffusion phenomenon during conventional and ultrasound extraction with the second Fick's law allowed the estimation of diffusivities and solvent penetration into the inner tissues and in meantime to accelerate the release of valuable metabolites.