## 4 Secondary Metabolites
### Extraction  
Pytochemical compounds from apples were extracted by using various extraction methods based on the application of different solvents and by heating and/or mixing. The most used techniques were Soxhlet extraction, maceration and hydro-distillation [60; 61; 62]. Conventional Soxhlet extraction still remains one of the most relevant approach to extract volatile compounds from apples [62]. The sample is placed in a thimble-holder, where it's slowly filled with condensed solvent from a distillation flask. Once the liquid has reached an overflow level, the whole contents of the thimble-holder is aspirated by a siphon, which unloads it back into the distillation flask, leaving the extracted analytes in the bulk liquid [45]. On the contrary, during maceration, the sample is immersed for a variable time in a solvent inside an airtight container in order to allow the analyte transferring [63]. Vacuum hydro-distillation, instead, uses water vapor to recover volatile and apolar components from fruit tissues, and, as Soxhlet, it has been mainly used for the extraction of apple aroma, because it gave the extracts closest to the fresh fruit [60]. The duration of the extraction process, and the large number of organic solvents used are the major drawbacks of these techniques. In fact, alternative approaches have emerged in an attempt to mitigate limitations of the conventional ones. The innovation is largely focused on finding technological solutions to diminish or even prevent the use of organic  
Figure 2: Main classes of polyphenols occurring in apples. solvents in extraction processes, in order to obtain more highly purified products containing fewer additional toxins [64]. The new methods include solid-liquid extractions (namely microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE), accelerated solvent extraction (ASE), supercritical fluid extraction (SFE)) and solid-phase microextraction (SPME) (Table 3). Organic solvents, such as hexane, acetone, methanol, ethanol or water, have been generally used under atmospheric pressure. The choice of the solvent largely depended on the polarity of the analytes. Extraction of polyphenols from apples has been carried out by using methods that differ for many variables, such as solvent, time, temperature and number of extractions; therefore, the data reported in the literature are not always easily comparable. According to [PERSON] et al. [65], the extraction of polyphenols from apples can easily be achieved via MAE, by using ethanol as extracting solvent, in a ratio to raw material of 22.9/1. Microwave-assisted extraction is based on the use of microwave energy to heat solvents in contact with a sample to allow the analytes partitioning from the sample matrix into the solvent [66]. Compared to Soxhlet and maceration, MAE results in a greater efficiency at a shorter time for the extraction of phenolic compounds from apple pomace. Moreover, accelerated operation can be regarded as a major advantage of MAE, which is useful especially at the industrial scale [61]. [PERSON] et al. [24] used a methanol extraction by adding pre-chilled MeOH (\(-20\)\({}^{\circ}\)C) to apple pulp and extracting at 70 \({}^{\circ}\)C in a homogenizer. The extract was then centrifuged and dried under nitrogen flow. This extraction method was not selective for polyphenols; in fact, it allowed the extraction of many hydrophilic metabolites. Furthermore, this protocol of extraction was found to be not very efficient for complete extraction of all apple polyphenols, because less polar molecules could remain inside the matrix, which is discarded. In order to avoid this problem, a multi-step extraction can be used [67; 68]. A first step involves the treatment of the dried apples cortex with hexane, to remove lipids, carotenoids and chlorophyll. Then, an extraction with methanol dissolves sugars, organic acids and phenolic compounds of low molecular weight. Lastly, the resulting residue is treated with aqueous acetone (4:6), in order to extract polymerized polyphenols. [PERSON] et al.