# Modern Techniques for Flavonoid Extraction--To Optimize or Not to Optimize?
## 4 Selection of Variables for the Optimization of Modern Techniques for Flavonoids Extraction  
### Ultrasound Assisted Extraction  
As described by [PERSON] et al. [64] ultrasound-assisted extraction (UAE) is considered an environmentally friendly extraction technique due to its high productivity with minimal solvent and time consumption and its suitability for molecules with high thermal sensitivity (Figure 4a). The frequency of ultrasound ranges from 20 kHz to 100 kHz, and high powers (10-1000 W/cm) can be generated, sufficient to break intermolecular bonds. When the energy is higher than 10 W/cm, a cavitation effect (growth and collapse of bubbles) occurs. When the bubble collapses near the cell wall, it generates high temperature (up to 5000 K) and high pressure (up to 100 ATM), allowing the solvent to penetrate the cell, resulting in enhanced mass transfer. In addition to mass transfer, high pressure and temperature can rupture the cell wall and cell membranes and reduce the particle size, releasing the intracellular material and promoting the extraction process [62; 63; 64]. Optimization of UAE extraction should include selection of the proper solvent, particle size, temperature, time and solvent-to-solid ratio, as in traditional methods, but also optimization of ultrasound parameters (power and frequency) [65; 66] (Figure 4b). Figure 4: (**a**) Scheme of ultrasound-assisted extraction. (**b**) Specific variables that should be optimized for UAE extraction. Figure 3: Common extraction variables that should be optimized for all extraction techniques. [PERSON] et al. [67] analyzed the effect of the ethanol concentration, particle size, solvent-to-solid ratio, temperature, ultrasonic power and ultrasonic frequency on the extraction of flavonoids from peanut shells and showed that usage of ultrasound reduces the extraction process from 280 to 80 min in the case of Soxhlet extraction. Similarly, [PERSON] et al. [68] studied the effect of methanol ratio, temperature and extraction time on the total flavonoid content of _Lobelia nicatinifolia_ leaves and showed that plant tissues were extensively ruptured during ultrasonication and that UAE significantly increased the antioxidant capacity of the extracts. [PERSON] et al. [69] compared the efficiency of conventional solid-liquid extraction and UAE for flavonoids extraction from grapefruit peels and showed that UAE was more than 50% more efficient regarding the bioactives extraction yield, while [PERSON] et al. [70] compared conventional extraction and UAE for the extraction of bioactive red Araca fruit (_Psidium cattleanium_ Sabine) and presented approximately 6% higher flavonoids extraction yield when using UAE. [PERSON] et al. [71] explored how to increase the yield of oleuppein, hydroxytyrosol, flavonoids and phenolic acids extracted from olive leaves in order to reduce operational expenses with the potential for using reduced solvent amount, shorter extraction periods, and reduced extraction temperatures. They showed that UAE with acetone as the extraction solvent can be efficiently used to produce olive leaves extracts that are rich in bioactive compounds. Moreover, [PERSON] et al. [72] proposed a negative pressure cavitation-based ultrasound-assisted extraction (NPC-UAE) for the efficient extraction of six flavonoids from the flower buds of _Sophora japonica_ L. using ethanol as the extraction solvent and showed that the combination of the two extraction methods resulted in better extraction yields compared to the UAE method, while [PERSON] et al. [73] coupled enzyme-assisted extraction with the UAE method to improve the extraction efficiency of bioactive compounds from _Gymmena sylvestre_. The proposed extraction method was found to be three times more efficient than the conventional maceration performed as a control. During ultrasound-assisted solvent enzymatic extraction (UAE-EAE) ultrasonic waves promote enzymatic extraction by cracking cell walls to allow for enzyme-aided reactions and the subsequent release of desired components [74,75]. [PERSON] et al.