9 Analytical Protocols in Antioxidant Capacity Measurement
211
acids (Crozier et al. 2009). The phenolic acids (non-flavonoid compounds) are generally composed of an aromatic ring attached to a carboxylic acid and hydroxyl groups,
they are divided into hydroxybenzoic acids (e.g., gallic acid) with the conformation
of C6-C1 and hydroxycinnamic acids (e.g., coumaric, ferulic, and caffeic acids) with
C6-C3 structures (Robbins 2003). Flavonoids, on the other hand, are characterized
by the structure C6-C3-C6, (e.g., quercetin and apigenin), i.e., two aromatic rings
linked by a central ring C3 (Panche et al. 2016). Their absorption spectra range from
280 to 370 nm for flavonoids and up to 320 nm for phenolic acids (Crozier et al.
2009).
The antioxidant mechanism of action involves a combined HAT from its hydroxyl
groups to the radical, forming a transition state of an OH bond with an electron (see
Fig. 9.4). Based on this, it is suggested that their ability to scavenge reactive species
is proportional to the number and position of hydroxyls. However, this antioxidant
capacity is significantly reduced when the reaction medium consists of a solvent
prone to the formation of hydrogen bonds as alcohol (Santos-Sánchez et al. 2019).
Additionally, the chromophore present in the structure of some phenolic
compounds, such as quercetin, may provide the ability to scavenge radicals via SET,
but may also act as chelators of metal ions, such as iron and copper, due to the
presence of 3’, 4’-hydroxyl groups in B ring, inhibiting the oxidation of low-density
lipoproteins (LDL) (Leopoldini et al. 2004).
9.3 Overview of Antioxidant Protocols
Natural extracts containing bioactive compounds are frequently evaluated for their
effectiveness by different in vitro chemical methods in variable reaction media.
Table 9.1 shows the relevant characteristics of the most cited protocols in the evaluation of microalgae antioxidants: DPPH, ABTS, FRAP, RC, ORAC-H, ORAC-L,
and PRSC.
Among non-competitive assays, DPPH is a colorimetric method, in which the
principle involves the scavenging of DPPH
• by antioxidants in a methanolic reaction
medium at pH 6.0, and the loss of absorption at 515 nm is monitored by spectrophotometry (Brandt-Willians and Berset 1995). The main mechanism involved is SET,
although HAT from the neutral antioxidant by DPPH was characterized as a marginal
reaction path due to its very slow reaction rate in strong hydrogen-bond-accepting
solvents, such as methanol and ethanol (Huang et al. 2005).
Unlike most reactive species, DPPH radical is characterized by its stability due to
the displacement of the spare electron on the molecule as a whole, so the molecule
does not dimerize (Alam et al. 2013). In its radical form, it has purple color resulting in
an absorption band at 515 nm that fades after reduction by an antioxidant compound
(assuming both radical and probe function) (see Fig. 9.5). Absorbance monitoring
occurs for approximately 30 min (time required for the reaction kinetics of most
compounds to reach steady state) (Santos-Sanchez et al. 2019). The whole reaction
takes place at room temperature (25 °C), there is no need of prior radical preparation
211
acids (Crozier et al. 2009). The phenolic acids (non-flavonoid compounds) are generally composed of an aromatic ring attached to a carboxylic acid and hydroxyl groups,
they are divided into hydroxybenzoic acids (e.g., gallic acid) with the conformation
of C6-C1 and hydroxycinnamic acids (e.g., coumaric, ferulic, and caffeic acids) with
C6-C3 structures (Robbins 2003). Flavonoids, on the other hand, are characterized
by the structure C6-C3-C6, (e.g., quercetin and apigenin), i.e., two aromatic rings
linked by a central ring C3 (Panche et al. 2016). Their absorption spectra range from
280 to 370 nm for flavonoids and up to 320 nm for phenolic acids (Crozier et al.
2009).
The antioxidant mechanism of action involves a combined HAT from its hydroxyl
groups to the radical, forming a transition state of an OH bond with an electron (see
Fig. 9.4). Based on this, it is suggested that their ability to scavenge reactive species
is proportional to the number and position of hydroxyls. However, this antioxidant
capacity is significantly reduced when the reaction medium consists of a solvent
prone to the formation of hydrogen bonds as alcohol (Santos-Sánchez et al. 2019).
Additionally, the chromophore present in the structure of some phenolic
compounds, such as quercetin, may provide the ability to scavenge radicals via SET,
but may also act as chelators of metal ions, such as iron and copper, due to the
presence of 3’, 4’-hydroxyl groups in B ring, inhibiting the oxidation of low-density
lipoproteins (LDL) (Leopoldini et al. 2004).
9.3 Overview of Antioxidant Protocols
Natural extracts containing bioactive compounds are frequently evaluated for their
effectiveness by different in vitro chemical methods in variable reaction media.
Table 9.1 shows the relevant characteristics of the most cited protocols in the evaluation of microalgae antioxidants: DPPH, ABTS, FRAP, RC, ORAC-H, ORAC-L,
and PRSC.
Among non-competitive assays, DPPH is a colorimetric method, in which the
principle involves the scavenging of DPPH
• by antioxidants in a methanolic reaction
medium at pH 6.0, and the loss of absorption at 515 nm is monitored by spectrophotometry (Brandt-Willians and Berset 1995). The main mechanism involved is SET,
although HAT from the neutral antioxidant by DPPH was characterized as a marginal
reaction path due to its very slow reaction rate in strong hydrogen-bond-accepting
solvents, such as methanol and ethanol (Huang et al. 2005).
Unlike most reactive species, DPPH radical is characterized by its stability due to
the displacement of the spare electron on the molecule as a whole, so the molecule
does not dimerize (Alam et al. 2013). In its radical form, it has purple color resulting in
an absorption band at 515 nm that fades after reduction by an antioxidant compound
(assuming both radical and probe function) (see Fig. 9.5). Absorbance monitoring
occurs for approximately 30 min (time required for the reaction kinetics of most
compounds to reach steady state) (Santos-Sanchez et al. 2019). The whole reaction
takes place at room temperature (25 °C), there is no need of prior radical preparation