## 3 Results and Discussion
### Investigations on the Effect of Ultrasound on the Structure of an Olive Leaf
#### 3.2.2 Histological Structure of the Leaf
These observations have never been reported, and the majority of published studies were performed on olives' leaves ground prior to extraction. Considering leaf surface observations and the histological structure of tissues stained with TBO, we can suggest two types of mechanisms occurring when ultrasound is applied: erosion and fragmentation (Figure 4) [17]. Observations point out that on nongrinded leaves, ultrasound acts mainly on the surface of the leaves; impacted structures are hairs and probably the cuticular layer of the adaxial surface of the leaves. Ultrasound tends to sever hairs, leaving their foot (probably rich in phenolic compounds given that they are strongly stained in blue by TBO) accessible to solvent and hence unveiling the cuticle beneath the hairs. This implies that ultrasound contributes to maximize the surface area of phenolic-containing structures and hence to enhance the accessibility of phenolics to the solvent. The effect of ultrasound was noticed after 5 min of sonication, but an increasing sonication duration (up to 60 min) led to the complete removal of hairs on the adaxial surface. On the abaxial surface, only a partial removal of hairs was noted after 60 min of sonication. Correlating these observations with polyphenol extraction, it could be concluded that the yield increase was probably due to a mechanical action of cavitation bubble implosion on the adaxial surface rather than on the abaxial surface although the latter was exposed to ultrasound. When applying power ultrasound, there are different effects like microstreaming, cavitation, a \"sponge effect\", and mechanical \"opening\" of canals in treated tissue [4]. Usually, researchers have reported the formation of free radicals, efficient release of compounds from intracellular space, faster drying, and faster freezing when using ultrasound as a pretreatment method. There is novel knowledge about macroscopic and microscopic effects of plant material. The authors studied the application of ultrasound in the extraction from  
Figure 4: Evidenced mechanisms of the effect of acoustic cavitation on leaf surface modification. -rosemary leaves, _Rosmarinus officinalis_ L. [37]. They suggested mechanisms and identify changes during ultrasound treatments of leaf structures. Ultrasound was compared to conventional processes, and the surfaces of untreated and treated leaves were examined by Scanning Electron Microscopy (SEM) and Environmental Scanning Electron Microscopy (ESEM). The authors also performed a cyto-histochemical study to analyze ultrasound-induced alterations on the inner structures. Considering macroscopic and microscopic investigation of leaf surface and cyto-histochemical study of tissues, we can suggest six types of mechanisms upon ultrasound treatment of plant tissue: erosion, shear forces, sonoporation, fragmentation, capillary effect, and detekuration. These mechanisms followed each other during ultrasound treatment, resulting in the gradual physical damage of tissues' structure. Another study analyzed the effect of ultrasound on almond skin cells. SEM analysis of treated almond skins showed a noticeable impact of ultrasound on skin cells [38]. In the study with an ultrasound bath, the optimization of ultrasound process parameters (time, frequency, and solvent) was studied to obtain high extraction yields, and the recovery of phlorotannins and total phenols from _Fucus vesiculusus_[39]. Another aim was to assess the damage caused by ultrasound on algal cell surfaces using SEM analysis. From SEM images, it was shown that the cell surface of the initial seaweed biomass was intact and surrounded by other impurities and residual materials, while the cell surfaces of the control samples (without ultrasound treatment) appeared to be smooth with an increased number of pores that allowed for diffusion of bioactive compounds to the media. At the end of these studies, we can see that the effect of ultrasound depends on the type of plant material studied and that there is no standard procedure [40].