2. Extractable polyphenols (EP)

The applied methodologies in the use of ARs to obtain EP depend on residue type and polyphenol stability. For example, acerola bagasse contains water [39], and if the extraction procedure is not done quickly, the residue will need to be dried to avoid microbiological contamination without affecting polyphenol stability. On the other hand, the probable water content of cocoa husk is low, and therefore, the polyphenols' extraction procedures are direct because it is a solid residue. Therefore, drying and extraction technologies or methodologies are necessary to obtain suitable yields of EP with proven antioxidant activity. The description of the drying and extraction methodologies of the EP will focus on the work with anthocyanidins because they are unstable compounds and the conditions of drying or extraction for anthocyanins are important to avoid their decomposition.

mixture to dissolve polar polyphenols. Then extraction with an organic solvent was done to recover less polar polyphenols. Anthocyanin yields found were better (5967–131, 868 mg/kg dm) [24] than those obtained from grape skins from fresh fruits (1211.8 mg/kg dm) [21] due to previous thermic and enzymatic treatments

Effects of temperature, time, and solvent concentration on polyphenol extraction from grape marc showed better extraction yields with the increase of water (30–50%) in the ethanol-water mixtures, maintaining the temperature at 60 °C for shorter periods (<8 h) to avoid polyphenol degradation [46]. Similar ethanol-water mixtures (40.4 and 55.4%) were used to extract the major components of grape cane: trans-resveratrol 19, trans-ε-viniferin 25, and ferulic acid 7 (Figure 2). However, a higher temperature (84 °C) was necessary to obtain the highest antioxidant activities (260.8 and 1378.7 μmol TE/g TEAC and ORAC methods) [47]. Anthocyanins extraction from grape skins, stems, and seeds was effective at 70 °C with ethanol-water mixtures, (1:1) and assisted with pulsed electric fields (PEF) (9 kV,

15 s), ultrasound (35 kHz, 70 °C, 1 h), and high hydrostatic pressurization

accelerated access of solvent (CO2/ethanol) to vegetal tissues, and TPC was

GAE/g dm [18].

71

(600 MPa, 70°C, 1 h). The extraction yields for PEF were 81 and 25% higher than those obtained by ultrasound and hydrostatic pressurization [48]. A combination of methods has also been used, such as the consecutive application of UAE (4 min, 80 °C, 20 kHz, 80 W) and SFE (8 MPa, 40 °C, CO2/ethanol) for polyphenols extraction from grape marc. The ultrasound treatment increased mass transfer and

increased 27% (2736 11 mg to 3493 61 GAE/100 g dw) [49]. SFE with different conditions (90% CO2, 5% ethanol, 5% water, 20 MPa, 40 °C) was more efficient than pressurized liquid extraction (PLE) in experiments with blueberry waste. Anthocyanidin yields were 808 0.1 mg/100 g and 248 0.2 mg/100 g for SFE and PLE (20 MPa, 50% water, pH 2, 40 °C) extractions, respectively [33]. PLE with increase of pressure and temperature and change of solvent (65 °C, 10 MPa, 75% ethanol) was shown to be more useful in the extraction of gallic acid 4 together with flavanones 41–45 from orange peels. The TPC obtained was 14.9 0.7 mg

Polyphenols different from anthocyanins, such as phenolic acids (147.4– 492.7 g/kg), gallic acid 4 (93.1–353.7 mg/kg), and flavonoids (2.52–13.5 mg/kg), were obtained with acidified methanol from residues of grape (Vitis vinifera L.) seed oil production [28], while protocatechuic acid 1 and chlorogenic acid 14 were extracted with hot water (92 3 °C, 2 min) [13]. Other solvents used for EP extraction are acetone/water mixtures (80%, 3 h stirring) to obtain flavonol 37, 40 glycosides and xanthones 17–18 from lyophilized mango peels (Table 1 and Figure 2) [31]. In general, EP extraction is made with water-organic solvents and assisted by conventional and nonconventional methods. However, when extraction conditions are severe as when heating above 70 °C, acid extraction or ultrasound exposure for long periods of time, then these conditions are also useful for obtaining

NEP, where the objective is promoting the breakdown of chemical bonds.

Non-extractable polyphenols (tannins and lignins) are low- or high-molecular-

weight compounds associated with vegetal tissue macromolecules; therefore, they are retained in the residue matrix during the extraction process. Depending on the monomeric structures and chemical reactivity of tannins, these are grouped in condensed and hydrolysable tannins. Condensed tannins are polyhydroxyflavan-3-ols oligomers and polymers linked by carbon-carbon bonds between flavanol

3. Non-extractable polyphenols (NEP)

during wine production, which helped release anthocyanins.

Antioxidant Compounds from Agro-Industrial Residue DOI: http://dx.doi.org/10.5772/intechopen.85184

#### 2.1 Waste drying

Valorization studies showed acerola bagasse (Malpighia emarginata DC) is a good source of antioxidants due to total phenol content (TPC), which ranges from 0.44 to 10.82 g gallic eq/100 g dm (dry matter) [39–41], while the contents of anthocyanins ascorbic acid and proanthocyanidins are 1.002 0.014, 1.002 0.014, and 0.7985 0.0213 g/100 g dw, respectively, and the antioxidant activity evaluation by DPPH• method showed 113.7 0.4 μmol trolox/g dw. However, this residue contains water and therefore must be dried for efficient handling. The drying of the residue has been carried out by hot air (60–80 °C, 4–6 m/s). This procedure showed moderate retention of phenolic (26–31%), anthocyanins (23–36%), and proanthocyanidins (21%) compounds [41]. Better results were obtained using a roto-aerated dryer (115 °C, 2.25 m/s) with a pretreatment of the sample (sprayed with ethanol), and total phenol compounds (TPC) increased 104.6% with respect to fresh residue [42]. A more recent drying method for acerola bagasse is dehydration in a thick-layer dryer, where drying was done at low temperatures (31.7 °C, 230 min, 0.4 m/s or 60 °C, 159.3 min, 0.4 m/s), which were enough to obtain a dry residue with TPC values similar to those obtained in fresh residue (2352.4 57.23 mg gallic acid/100 g dm) [42]. Drying studies of other ARs are described for grape and olive waste using thin-layer drying (air temperature 20–110 °C) [43, 44], and sustainable drying strategies such as the use of solar dryers have been described [45].

#### 2.2 ARs extraction

Generally, EP extraction procedures are done using mixtures of water-organic solvents and assisted by microwave (MAE), heating, and ultrasound (UAE). In recent years, pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) have been applied, which could be better options because polyphenols are not exposed to severe conditions that promote degradation reactions. In addition, temperature control is a common method to assist the extraction procedures. For example, anthocyanidins, procyanidins, and flavonols were obtained from grape skins using MAE and UAE at 50 5 °C. This work also demonstrated that the yields obtained by MAE with UAE were improved up to 40% (86.39–121.18 mg/100 g dm) [21]. Acid conditions have also been tested to improve the extraction of anthocyanins (anthocyanidin glycosides) from grape peels separated from red grape pomace from vintages 2001 to 2002. The extraction was made in two steps. First, the residue was macerated (2 h) with methanol/HCl 0.1 (v/v) with oxygen reduced in the

#### Antioxidant Compounds from Agro-Industrial Residue DOI: http://dx.doi.org/10.5772/intechopen.85184

2. Extractable polyphenols (EP)

2.1 Waste drying

Antioxidants

dryers have been described [45].

2.2 ARs extraction

70

The applied methodologies in the use of ARs to obtain EP depend on residue type and polyphenol stability. For example, acerola bagasse contains water [39], and if the extraction procedure is not done quickly, the residue will need to be dried to avoid microbiological contamination without affecting polyphenol stability. On the other hand, the probable water content of cocoa husk is low, and therefore, the

polyphenols' extraction procedures are direct because it is a solid residue. Therefore, drying and extraction technologies or methodologies are necessary to obtain suitable yields of EP with proven antioxidant activity. The description of the

drying and extraction methodologies of the EP will focus on the work with

or extraction for anthocyanins are important to avoid their decomposition.

anthocyanins ascorbic acid and proanthocyanidins are 1.002 0.014,

anthocyanidins because they are unstable compounds and the conditions of drying

Valorization studies showed acerola bagasse (Malpighia emarginata DC) is a good source of antioxidants due to total phenol content (TPC), which ranges from 0.44 to 10.82 g gallic eq/100 g dm (dry matter) [39–41], while the contents of

1.002 0.014, and 0.7985 0.0213 g/100 g dw, respectively, and the antioxidant activity evaluation by DPPH• method showed 113.7 0.4 μmol trolox/g dw. However, this residue contains water and therefore must be dried for efficient handling. The drying of the residue has been carried out by hot air (60–80 °C, 4–6 m/s). This procedure showed moderate retention of phenolic (26–31%), anthocyanins (23–36%), and proanthocyanidins (21%) compounds [41]. Better results were obtained using a roto-aerated dryer (115 °C, 2.25 m/s) with a pretreatment of the sample (sprayed with ethanol), and total phenol compounds (TPC) increased 104.6% with respect to fresh residue [42]. A more recent drying method for acerola bagasse is dehydration in a thick-layer dryer, where drying was done at low temperatures (31.7 °C, 230 min, 0.4 m/s or 60 °C, 159.3 min, 0.4 m/s), which were enough to obtain a dry residue with TPC values similar to those obtained in fresh residue (2352.4 57.23 mg gallic acid/100 g dm) [42]. Drying studies of other ARs are described for grape and olive waste using thin-layer drying (air temperature 20–110 °C) [43, 44], and sustainable drying strategies such as the use of solar

Generally, EP extraction procedures are done using mixtures of water-organic solvents and assisted by microwave (MAE), heating, and ultrasound (UAE). In recent years, pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) have been applied, which could be better options because polyphenols are not exposed to severe conditions that promote degradation reactions. In addition, temperature control is a common method to assist the extraction procedures. For example, anthocyanidins, procyanidins, and flavonols were obtained from grape skins using MAE and UAE at 50 5 °C. This work also demonstrated that the yields obtained by MAE with UAE were improved up to 40% (86.39–121.18 mg/100 g dm) [21]. Acid conditions have also been tested to improve the extraction of anthocyanins (anthocyanidin glycosides) from grape peels separated from red grape pomace from vintages 2001 to 2002. The extraction was made in two steps. First, the residue was macerated (2 h) with methanol/HCl 0.1 (v/v) with oxygen reduced in the

mixture to dissolve polar polyphenols. Then extraction with an organic solvent was done to recover less polar polyphenols. Anthocyanin yields found were better (5967–131, 868 mg/kg dm) [24] than those obtained from grape skins from fresh fruits (1211.8 mg/kg dm) [21] due to previous thermic and enzymatic treatments during wine production, which helped release anthocyanins.

Effects of temperature, time, and solvent concentration on polyphenol extraction from grape marc showed better extraction yields with the increase of water (30–50%) in the ethanol-water mixtures, maintaining the temperature at 60 °C for shorter periods (<8 h) to avoid polyphenol degradation [46]. Similar ethanol-water mixtures (40.4 and 55.4%) were used to extract the major components of grape cane: trans-resveratrol 19, trans-ε-viniferin 25, and ferulic acid 7 (Figure 2). However, a higher temperature (84 °C) was necessary to obtain the highest antioxidant activities (260.8 and 1378.7 μmol TE/g TEAC and ORAC methods) [47]. Anthocyanins extraction from grape skins, stems, and seeds was effective at 70 °C with ethanol-water mixtures, (1:1) and assisted with pulsed electric fields (PEF) (9 kV, 15 s), ultrasound (35 kHz, 70 °C, 1 h), and high hydrostatic pressurization (600 MPa, 70°C, 1 h). The extraction yields for PEF were 81 and 25% higher than those obtained by ultrasound and hydrostatic pressurization [48]. A combination of methods has also been used, such as the consecutive application of UAE (4 min, 80 °C, 20 kHz, 80 W) and SFE (8 MPa, 40 °C, CO2/ethanol) for polyphenols extraction from grape marc. The ultrasound treatment increased mass transfer and accelerated access of solvent (CO2/ethanol) to vegetal tissues, and TPC was increased 27% (2736 11 mg to 3493 61 GAE/100 g dw) [49]. SFE with different conditions (90% CO2, 5% ethanol, 5% water, 20 MPa, 40 °C) was more efficient than pressurized liquid extraction (PLE) in experiments with blueberry waste. Anthocyanidin yields were 808 0.1 mg/100 g and 248 0.2 mg/100 g for SFE and PLE (20 MPa, 50% water, pH 2, 40 °C) extractions, respectively [33]. PLE with increase of pressure and temperature and change of solvent (65 °C, 10 MPa, 75% ethanol) was shown to be more useful in the extraction of gallic acid 4 together with flavanones 41–45 from orange peels. The TPC obtained was 14.9 0.7 mg GAE/g dm [18].

Polyphenols different from anthocyanins, such as phenolic acids (147.4– 492.7 g/kg), gallic acid 4 (93.1–353.7 mg/kg), and flavonoids (2.52–13.5 mg/kg), were obtained with acidified methanol from residues of grape (Vitis vinifera L.) seed oil production [28], while protocatechuic acid 1 and chlorogenic acid 14 were extracted with hot water (92 3 °C, 2 min) [13]. Other solvents used for EP extraction are acetone/water mixtures (80%, 3 h stirring) to obtain flavonol 37, 40 glycosides and xanthones 17–18 from lyophilized mango peels (Table 1 and Figure 2) [31]. In general, EP extraction is made with water-organic solvents and assisted by conventional and nonconventional methods. However, when extraction conditions are severe as when heating above 70 °C, acid extraction or ultrasound exposure for long periods of time, then these conditions are also useful for obtaining NEP, where the objective is promoting the breakdown of chemical bonds.

#### 3. Non-extractable polyphenols (NEP)

Non-extractable polyphenols (tannins and lignins) are low- or high-molecularweight compounds associated with vegetal tissue macromolecules; therefore, they are retained in the residue matrix during the extraction process. Depending on the monomeric structures and chemical reactivity of tannins, these are grouped in condensed and hydrolysable tannins. Condensed tannins are polyhydroxyflavan-3-ols oligomers and polymers linked by carbon-carbon bonds between flavanol

units. These are also known as proanthocyanidins because the butanol/HCl/heat treatment produces a red anthocyanidin [54]. Hydrolysable tannins are multiple esters of gallic acid with glucose and products of oxidative reactions, and they can be soluble (EP) or non-soluble (NEP) (Figure 3) [55]. Lignin is a phenylpropanoid (C6C3) polyphenol where the monomeric units are p-coumaryl 71, coniferyl, and sinapyl. Occurrence of monomeric units in lignin varies according to the taxonomic origin of the ARs, e.g., gymnosperms or angiosperms [56].

laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS), LC LC coupled to tandem mass spectrometry, pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS), and nuclear magnetic resonance (NMR), which has been key to making detailed chemical studies of high-molecular-weight polyphenols [36, 64–66]. In the following paragraphs, some examples of chemical studies of NEP are presented to give an overview of the structural complexity that exists in them.

Antioxidant Compounds from Agro-Industrial Residue DOI: http://dx.doi.org/10.5772/intechopen.85184

Studies in pomegranate by-products led to the identification of several polyphenols. EP was extracted with acetone 70% (ultrasound-assisted 20 min, 30 °C), and NEP was previously subjected to basic hydrolysis of insoluble residues before extraction under the same conditions. Ellagic acid 6 and monogalloyl-hexoside 57 were the main compounds, in addition to ellagic acid derivates, valoneic acid bilactone I and II 58, punicalagin 59 and isomers, trigalloylglucopiranose I and II, and granatin, among 34 hydrolysable tannins described in Figure 4. Moreover, condensed tannins such as procyanidin dimers 30 and gallocatechin (or catechin) hexoside were described. Antioxidant activity for polyphenol (EP and NEP) fractions was 0.12–3.58 mmol TE/g dm (DPPH) and 5.34–208.73 μmol TE/g dm (ABTS radical-scavenging activity) [66]. Oligomeric proanthocyanidins such as dimer, trimer, tetramer, pentamer, and hexamer units (MW 600, 889, 1177, 1465, and 1753 amu) were identified in coffee pulp by MALDI-TOF-MS analysis. Extraction of these tannins was made with acid aqueous acetone [67]. Acetone/water also was used to extract anthocyanidins from litchi pericarp (Litchi chinensis), after its incubation with Aspergillus awamori. Total extractable tannin recovery was increased up to 59%, and ESI-TOF-MS analysis revealed A. awamori degraded B-type condensed

tannin and showed low capacity to degrade the condensed tannin A-type.

3.1 Tannins

Figure 4.

73

Hydrolysable tannins identified in pomegranate by-products [55, 69].

Non-extractable polyphenols are common in almost all ARs and represent significant polyphenol percentages of total phenol content (TPC) in vegetal tissues. For example, NEP quantification in peels from apple, banana, kiwi, mandarin, mango, nectarine, orange, pear, and watermelon showed that, of the total phenols found, 7–82% correspond to NEP [58]. Currently research in appropriate methodologies for the extraction of NEP is a priority topic because of the economic advantages of the use of ARs. Some examples of research on the best conditions for the extraction of NEP from ARs are those for the use of cocoa by-products, which involve extractions assisted by ultrasound [20, 35], thermic treatment [59, 60], hydrodynamic cavitation [35], pressurized liquids [50], pulsed electric field [61], subcritical water hydrolysis [62], and solid fermentation [63]. There are also reports on detailed chemical studies of the NEP structure thanks to modern analytical instrumentation, such as liquid chromatography (LC) coupled to matrix-assisted

Figure 3.

Structures of condensed tannins (proanthocyanidins), hydrolysable tannins (gallotannins), and lignin (5-hydroxyguaiacyl residue) [55, 57].

laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS), LC LC coupled to tandem mass spectrometry, pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS), and nuclear magnetic resonance (NMR), which has been key to making detailed chemical studies of high-molecular-weight polyphenols [36, 64–66]. In the following paragraphs, some examples of chemical studies of NEP are presented to give an overview of the structural complexity that exists in them.
