## 5 Extraction Methods  
To date, little information exists about the optimization of MAA extraction in macrosidea at a significant scale. Most authors extracted MAAs by maceration or sonication in distilled water, ethanol, methanol, or different percentages of methanol/ethanol:water and incubation at low temperature. Some authors compared different solvents and obtained a higher extraction with combinations of methanol/ethanol:water, in comparison with only methanol or ethanol extractions [48; 72; 77; 89; 90]. Conventional extraction methods included different cell disruption techniques: (1) mechanical (e.g., mortar and pestle or blender type instrument); (2) thermal (e.g., water bath or oven); and (3) chemical (e.g., acid or alkaline solution). The combination of these methods can increase the yield of the extraction. The type of extraction and the solvent used will be selected depending on the physico-chemical characteristics of the molecule of interest and the purpose for its extraction. For cosmeceutical applications, MAA extraction should be undertaken with so-called green solvents, such as water and ethanol, with low or no toxicity [91; 92]. In recent years, novel extraction methods have been developed in an attempt to minimize or eliminate the use of non-renewable and polluting solvents in the extraction process of bioactive compounds. As a result, the use of green solvents has increased, leading to higher quality products, decreasing the formation of by-products, improving the separation of products, and avoiding toxicity [93]. Some authors reviewed the different existing novel extraction techniques that can increase the yield and reduce the time of extraction [94; 95; 96]; some of these could be used for MAA extraction due to the application of polar and non-toxic solvents:  
Enzyme-assisted extraction (EAE): This method is based on the high specificity and selectivity of enzymes in aqueous solutions. Enzymes (such as carrageenanases, agarases, or proteases) can degrade the cell wall, releasing the inside bioactive compounds. This technique works under moderate conditions, protecting the molecules of interest from high temperature or acidic/alkaline pH. A disadvantage is the cost of the enzymes, which makes it to scale up the process. EAE has been used in marine organisms to obtain different bioactive compounds, such as polyphenols, fucoidans, carotenoids, and phycobiliproteins [97; 98; 99; 100; 101; 102]. Ultrasound-assisted extraction (UAE): This technique uses acoustic waves (20-100 kHz) that travel through the solvent producing cavitation bubbles that can cause cell disruptions. It can be used with a wide variety of solvents, and the temperature during the extraction is relatively low (ice can be used to reduce the temperature). The equipment needed for this extraction is not very expensive, meaning it can be used for large-scale applications. Some studies have indicated that the combination of EAE and UAE generated a synergistic effect, increasing the extraction percentage from 74% to 91%. UAE has been used by different authors to extract polyphenols, polysaccharides, or phycobiliproteins, among others [99; 103; 104]. Microwave-assisted extraction (MAE): Microwaves (non-ionizing electromagnetic radiation; 300 MHz-300 GHz) induce vibration of water molecules inside the cell by interacting with the dipole structure of the water molecule. MAE is a cost-effective technique that can use lower solvent amounts and can be combined with other techniques such as UAE. A disadvantage is that MAE is not suitable for heat-sensitive biocompounds. MAE has been used mainly for polyphenol and fucoidan extraction [105; 106]. Pressurized liquid extraction (PLE): This technique is performed at high temperature (50-200 \({}^{\circ}\)C) and high pressures (10-15 MPa). The main consequences of PLE utilization are increasing mass transfer, and decreasing the surface tension of solvents and viscosity.