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Comparing the four spectrophotometric assays DPPH, ABTS, FRAP and Folin-
Ciocalteu to evaluate the antioxidant activity of a set of lignins

Conference Paper · March 2023

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Jessica Rumpf René Burger

University of Bonn Hochschule Bonn-Rhein-Sieg
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Margit Schulze
Rheinische Friedrich Wilhelms University Bonn
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 PAPER ID: 3818303
DIVISION: CELL
SESSION TYPE: Oral - In-person

Comparative study of the antioxidant activity and total phenol content of organosolv lignins
from various renewable resources using different spectrophotometric assays
Jessica Rumpf 1,2, René Burger 1, and Margit Schulze 1,2*

1
Department of Natural Sciences, Bonn-Rhein-Sieg University of Applied Sciences, Germany
2
Agricultural Faculty, Rheinische Friedrich-Wilhelms-University Bonn, Germany

Antioxidants play a crucial role in human health, as they can prevent oxidative stress related diseases like
high blood pressure, neurodegenerative disorders, or cancer. In addition, natural antioxidants are getting
more and more attention due to the progressive increase of petrochemical consumption. One key topic in
biorefinery research is lignin, a polyphenolic biopolymer that can be extracted from various biomasses.
The great number of phenolic hydroxyl groups is one reason for its antioxidant activity (AOA), as they are
able to end oxidative chain reactions and quench free radicals. However, due to this molar complexity, the
investigation of its AOA is sophisticated, as no single method is capable to measure all natural reactions
occurring in vivo, and thus can depict its whole potential.

In this study, four different assays, namely the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS), Ferric ion Reducing Antioxidant Potential (FRAP) and Folin
Ciocalteu (FC) assay, respectively, were used to investigate the AOA of 50 organosolv lignins from
Miscanthus, Paulownia, Silphium, beech, and wheat straw, and two kraft lignins.

The coefficient of variation (CV) was determined for each method and was lowest for ABTS (0.25 %) and
highest for DPPH (5.30 %). When comparing these two radical scavenging assays, ABTS seems to be
favorable regarding reaction time, sensitivity, and reproducibility. The best correlation between two
assays is found for FRAP and FC (R² = 0.944), which is not surprising as both rely on the same mechanism,
the single electron transfer (SET). In contrast, the underlying reaction mechanisms for ABTS and DPPH
cannot be assigned to either SET or hydrogen atom transfer (HAT) but are more complex as both occur in
varying proportions. Nevertheless, also a good correlation (R² > 0.75) between ABTS and FRAP and FC,
respectively, could be found, while DPPH merely correlates with the others (R² < 0.5), leading to the
conclusion that the DPPH assay is closer to the HAT mechanism. Conversely, this does not mean that the
DPPH assay is not feasible for the measurement of lignins, but rather that it reflects varying antioxidative
attributes, which might be due to a different reaction mechanism.

Rumpf, J.; Burger, R.; Schulze, M. Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to
assess the antioxidant capacity of lignins. Int. J. Biol. Macromol. 2023, doi:10.1016/j.ijbiomac.2023.123470

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