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T. Casagrande do Nascimento et al.
9.1 Introduction
Over the past few years, the need to discover sources of bioactive compounds with
potential to scavenge reactive chemical species has become increasingly common, as
these reactive species induce oxidative damages, from technological losses in food
matrices to structural modifications in biological components, causing irreversible
damage to human health (Ahmed et al. 2014; Ayala et al. 2014; Gaschler and Stockwell 2017). Oxidative stress has been strongly associated with numerous chronic
degenerative diseases and ageing processes (Bhat et al. 2015; Peña-Bautista et al.
2019), which justifies the intensified search for antioxidant compounds capable of
delaying or inhibiting these oxidative processes.
In this context, microalgae (including cyanobacteria) have become the target of
much research as they are recognized for producing diverse biologically active
compounds including carotenoids, chlorophylls, phycobiliproteins, and phenolic
compounds (Borowitzka 2018; Goiris et al. 2015; Hossain et al. 2016; Jacob-Lopes
et al. 2019). Carotenoids, chlorophylls, and phycobiliproteins are the main classes
of microalgae pigments (Fernandes et al. 2017; Patias et al. 2017; Rodrigues et al.
2015; Zepka et al. 2019; Jacob-Lopes et al. 2019). On the other hand, although some
colored flavonoids have been reported in microalgae, the most common phenolic
compounds are colorless and structurally acidic (Mahmood et al. 2019; Goiris et al.
2014). These bioactive compounds have in common the ability to scavenge reactive oxygen species (ROS), and their antioxidant potential is mostly related to their
structures.
The first step in investigating the antioxidant potential of these compounds
occurs via in vitro methodologies. Approximately 19 in vitro assays are used
for antioxidant evaluation (Alam and Bristi 2013). Among them, the most relevant for the assessment of microalgae extracts include ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH, (2,2-diphenyl-1-picrylhydrazyl),
FRAP (Ferric Reducing Antioxidant Power), RC (Reducing Capacity), ORACH (Oxygen Radical Absorbance Capacity for hydrophilic antioxidants), ORACL (Oxygen Radical Absorbance Capacity for lipophilic antioxidants), and PRSC
(Peroxyl Radical Scavenging Capacity) (Brand-Williams et al.,1995; Benzie and
Strain 1996; Ou et al. 2001; Re et al. 1999; Rodrigues et al. 2012).
In general, the objective of these assays is to promote the contact of the antioxidant compound (isolated or present in extracts) with some kind of reactive species
under specific reaction conditions that allows measuring directly or indirectly the
antioxidant capacity in comparison with a reference standard (Alam and Bristi 2013).
Although the antioxidant assays serve the same purpose to estimate the antioxidant capacity, they have different reaction media, which may be competitive or
not during the mechanisms of electron transfer (Single-Electron Transfer, SET) or
a hydrogen atom (Hydrogen Atom Transfer, HAT). While in ORAC-H, ORAC-L,
and PRSC assays there is a competition between an antioxidant and an oxidizable
substrate (probe), in ABTS, FRAP, DPPH assays, and RC this competition does not
happen (Apak et al. 2016; Huang et al. 2005). The competition characteristic of
T. Casagrande do Nascimento et al.
9.1 Introduction
Over the past few years, the need to discover sources of bioactive compounds with
potential to scavenge reactive chemical species has become increasingly common, as
these reactive species induce oxidative damages, from technological losses in food
matrices to structural modifications in biological components, causing irreversible
damage to human health (Ahmed et al. 2014; Ayala et al. 2014; Gaschler and Stockwell 2017). Oxidative stress has been strongly associated with numerous chronic
degenerative diseases and ageing processes (Bhat et al. 2015; Peña-Bautista et al.
2019), which justifies the intensified search for antioxidant compounds capable of
delaying or inhibiting these oxidative processes.
In this context, microalgae (including cyanobacteria) have become the target of
much research as they are recognized for producing diverse biologically active
compounds including carotenoids, chlorophylls, phycobiliproteins, and phenolic
compounds (Borowitzka 2018; Goiris et al. 2015; Hossain et al. 2016; Jacob-Lopes
et al. 2019). Carotenoids, chlorophylls, and phycobiliproteins are the main classes
of microalgae pigments (Fernandes et al. 2017; Patias et al. 2017; Rodrigues et al.
2015; Zepka et al. 2019; Jacob-Lopes et al. 2019). On the other hand, although some
colored flavonoids have been reported in microalgae, the most common phenolic
compounds are colorless and structurally acidic (Mahmood et al. 2019; Goiris et al.
2014). These bioactive compounds have in common the ability to scavenge reactive oxygen species (ROS), and their antioxidant potential is mostly related to their
structures.
The first step in investigating the antioxidant potential of these compounds
occurs via in vitro methodologies. Approximately 19 in vitro assays are used
for antioxidant evaluation (Alam and Bristi 2013). Among them, the most relevant for the assessment of microalgae extracts include ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH, (2,2-diphenyl-1-picrylhydrazyl),
FRAP (Ferric Reducing Antioxidant Power), RC (Reducing Capacity), ORACH (Oxygen Radical Absorbance Capacity for hydrophilic antioxidants), ORACL (Oxygen Radical Absorbance Capacity for lipophilic antioxidants), and PRSC
(Peroxyl Radical Scavenging Capacity) (Brand-Williams et al.,1995; Benzie and
Strain 1996; Ou et al. 2001; Re et al. 1999; Rodrigues et al. 2012).
In general, the objective of these assays is to promote the contact of the antioxidant compound (isolated or present in extracts) with some kind of reactive species
under specific reaction conditions that allows measuring directly or indirectly the
antioxidant capacity in comparison with a reference standard (Alam and Bristi 2013).
Although the antioxidant assays serve the same purpose to estimate the antioxidant capacity, they have different reaction media, which may be competitive or
not during the mechanisms of electron transfer (Single-Electron Transfer, SET) or
a hydrogen atom (Hydrogen Atom Transfer, HAT). While in ORAC-H, ORAC-L,
and PRSC assays there is a competition between an antioxidant and an oxidizable
substrate (probe), in ABTS, FRAP, DPPH assays, and RC this competition does not
happen (Apak et al. 2016; Huang et al. 2005). The competition characteristic of