292
absorbance, results are calibrated with ascorbic acid or trolox (μmol per g of dry
matter). The ORAC, oxygen radical absorbance capacity, assay is based on inhibition of the activity of reactive species by an antioxidant, resulting in loss of emission
of fluorescence of phycoerythrin or fluorescein (Galili and Hovav 2014). In contrast
to the other assays, the ORAC method combines both inhibition time and degree of
inhibition expressed as a single quantity. This assay measures the kinetics of the
decrease in fluorescence for each sample compared to a blank by plotting fluorescence emission versus time. ORAC values are generally given as trolox equivalents.
Widely used until 2012, this assay has been withdrawn as classical antioxidant
assay because there is no physiological proof that ORAC values have any true biological significance (Schaich et al. 2015). Other biological studies requiring expensive kits or cell culture could be employed, as well as pulse radiolysis (Quideau
et al. 2011) or electrochemical (René et al. 2010) techniques. But the majority of
studies have reported antioxidant activity through assays described above due to
their relative simplicity, notably DPPH and ABTS
•+
.
As a result, they are similarly widely employed with deep eutectic solvent extraction. As an illustration, we could quote the extraction of microalgae polyphenols in
polyol-based deep eutectic solvent (Wan Mahmood et al. 2019). The extracts
obtained in conventional solvents (ethyl acetate and water) gave much lower antioxidant activity (DPPH assay) than those provided by deep eutectic solvent (Wan
Mahmood et al. 2019). The deep eutectic solvent extracts presenting the best antioxidant activity were also characterized by the largest total phenolic contents as
determined by Folin-Ciocalteu (Wan Mahmood et al. 2019). It is then tempting to
directly correlate the high content of phenolic compounds to the high antioxidant
activity.
However, it is interesting to consider another example: the extraction of phenolic
compounds from onion peels in deep eutectic solvent based on choline chloride
associated with urea and water (1:2:4), sucrose and water (4:1:8), and D-sorbitol
and water (3:1:10). Ferric reducing-antioxidant power (FRAP) was almost five
times higher for extract in choline chloride:urea:water than in conventional aqueous
methanol or water extract, while the two other deep eutectic solvents gave twice the
values. In contrast, onion peel extracts in aqueous methanol or in choline
chloride:sorbitol:water were found to have a better antioxidant activity in terms of
DPPH assay. The DPPH trend does not fully correlate with the total phenolic content (lowest value for extraction with choline chloride:sorbitol:water).
The nature of the antioxidant activity mechanism (electron transfer or radical
scavenging) may be different according to the solvents used for extraction, which
remained in the extracts, possibly explaining these different results.
In another example, Jeong et al. have evaluated the total phenolic and flavonoid
contents of peppermint leaves (Mentha piperita) extracted notably with choline
chloride:glucose (5:2) (Jeong et al. 2018). These values were obtained with the classical colorimetric assays. They found a high correlation (r > 0.9; p < 0.05) of the
amount of phenolic and flavonoid compounds with the antioxidant activity, when
L. Percevault et al.
absorbance, results are calibrated with ascorbic acid or trolox (μmol per g of dry
matter). The ORAC, oxygen radical absorbance capacity, assay is based on inhibition of the activity of reactive species by an antioxidant, resulting in loss of emission
of fluorescence of phycoerythrin or fluorescein (Galili and Hovav 2014). In contrast
to the other assays, the ORAC method combines both inhibition time and degree of
inhibition expressed as a single quantity. This assay measures the kinetics of the
decrease in fluorescence for each sample compared to a blank by plotting fluorescence emission versus time. ORAC values are generally given as trolox equivalents.
Widely used until 2012, this assay has been withdrawn as classical antioxidant
assay because there is no physiological proof that ORAC values have any true biological significance (Schaich et al. 2015). Other biological studies requiring expensive kits or cell culture could be employed, as well as pulse radiolysis (Quideau
et al. 2011) or electrochemical (René et al. 2010) techniques. But the majority of
studies have reported antioxidant activity through assays described above due to
their relative simplicity, notably DPPH and ABTS
•+
.
As a result, they are similarly widely employed with deep eutectic solvent extraction. As an illustration, we could quote the extraction of microalgae polyphenols in
polyol-based deep eutectic solvent (Wan Mahmood et al. 2019). The extracts
obtained in conventional solvents (ethyl acetate and water) gave much lower antioxidant activity (DPPH assay) than those provided by deep eutectic solvent (Wan
Mahmood et al. 2019). The deep eutectic solvent extracts presenting the best antioxidant activity were also characterized by the largest total phenolic contents as
determined by Folin-Ciocalteu (Wan Mahmood et al. 2019). It is then tempting to
directly correlate the high content of phenolic compounds to the high antioxidant
activity.
However, it is interesting to consider another example: the extraction of phenolic
compounds from onion peels in deep eutectic solvent based on choline chloride
associated with urea and water (1:2:4), sucrose and water (4:1:8), and D-sorbitol
and water (3:1:10). Ferric reducing-antioxidant power (FRAP) was almost five
times higher for extract in choline chloride:urea:water than in conventional aqueous
methanol or water extract, while the two other deep eutectic solvents gave twice the
values. In contrast, onion peel extracts in aqueous methanol or in choline
chloride:sorbitol:water were found to have a better antioxidant activity in terms of
DPPH assay. The DPPH trend does not fully correlate with the total phenolic content (lowest value for extraction with choline chloride:sorbitol:water).
The nature of the antioxidant activity mechanism (electron transfer or radical
scavenging) may be different according to the solvents used for extraction, which
remained in the extracts, possibly explaining these different results.
In another example, Jeong et al. have evaluated the total phenolic and flavonoid
contents of peppermint leaves (Mentha piperita) extracted notably with choline
chloride:glucose (5:2) (Jeong et al. 2018). These values were obtained with the classical colorimetric assays. They found a high correlation (r > 0.9; p < 0.05) of the
amount of phenolic and flavonoid compounds with the antioxidant activity, when
L. Percevault et al.