Probing natural phenolic compounds by EPR spectroscopy – a prooxidant assay

Cezara Zagrean-Tuza1,2, Augustin Mot1,2, Tomasz Chemiel3, Atilla Bende1, Ioan Turcu1

1National Institute for Research and Development of Isotopic and Molecular Technology, Cluj-

Napoca, Romania2Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, Cluj-Napoca,

Romania3Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland

King Midas of Phrygia – the golden touch

https://industryreports24.com/522862/global-food-antioxidants-market-top-manufactures-regions-trends-analysis-types-market-size-

and-forecasts-2019-2027/

“Golden” antioxidants

Natural phenolic compounds – “golden” antioxidants

flavonoids

proanthocyanidins

hydroxybenzoic acids

cinnamic acids xanthonoids

calchonoids

coumarins anthocyanidins

semiquinones quinones

Es-Safi N.E., et al., Molecules, 12 (2007) 2228-2258

They fight against free radicals…

HO●HOO●

O2●-

RO●

ROO●

H2O2

1O2

Ph-OH + R●→ Ph-O● + RH

ROS – reactive oxygen species

By generating other free radicals…

Sakihama Y. et al., Toxicology 177 (2002) 67–80

Other mechanisms of prooxidant reactivity:

• direct prooxidant action

• Inhibition of mitochondrial respiration

• oxidation of other endogenous antioxidants

• DNA damage

Procházková D. et al., Fitoterapia 82 (2011) 513–523

Ph-OH + R●→ Ph-O● + RH

Antioxidant polyphenols in cancer

treatment: Friend, foe or foil?

Seminars in Cancer Biology 46 (2017) 1–13

Probing the “golden” antioxidants. Analytical assessment…

An assay based on laccases – “clean phenoxyl generator”

H64

H398H400

H66

H109

H454

H452

H111

H458

H395

C453

I455

T1CuT3Cu

T3Cu

T2Cu

12.9 Å

2.78

Substrate oxidation site

Oxygen reduction site

Ph-OH

Ph-O●

O2

2H2O

4H+

0

0.3

0.6

0.9

1.2

1.5

1.8

450 500 550 600 650 700 750 800

wavelength (nm)

Abs

0.5

0.8

1.1

1.4

0 2 4 6 8

Reaction time (min)

Abs (

577 n

m)

oxyHb laccase

quercetin

quercetin

quercetin

Oxy-hemoglobin, our life sustaining protein…

Hb-FeII Hb-FeIII

oxy form

met form

1.5

1.8

2.1

2.4

2.7

3

3.3

-2.1 -0.1 1.9

ln[quercetin (uM)]A

UC

(uM

x m

in)

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4

Time (min)

Rel

ativ

e ab

s (5

77 n

m)

0.125 µM

0.25 µM

0.50 µM

1 µM

2 µM4 µM

6 µM

Quantification of pro-oxidant activity

Mean

Mean±S

E

Mean±S

D

Nasasel

Sarmas

Gledin

Pestera

Livada

Carei

Cristian

Orlat

Brebi

Magura

Fagaras

Romuli

Gura Raului

Pastoral

10

12

14

16

18

20

22

24

26

28

30

Prooxidant quercetin factor (umol/mg sample)

Pro

oxid

an

t activ

ity fo

r pro

polis

sam

ples fro

m d

ifferent flo

ral o

rigin

EPR spectroscopy detection of antioxidants-based radicals

Our X band Bruker EPR spectrometer

In capillaries…

3500 3505 3510 3515 3520 3525

Gauss

3500 3510 3520 3530Gauss

quercetinhyperoside

quercitrin

rutinisoquercitrin

EPR spectroscopy detection of antioxidants-based radicals

caffeic acid

chlorogenic acid

rosmarinic acid

elagic acid

Phytother. Res. 30: 1379–1391 (2016)

Our new assay: ROS generation by “golden” antioxidants

“golden” antioxidants ROS species

How much prooxidant? Could you quantify? Yes!

y = 2,3034x + 0,5877R² = 0,9998

0

100

200

300

400

500

600

0,00 50,00 100,00 150,00 200,00 250,00

dd

Inte

gra

l

[Spin] µM

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

18,00

20,00

3440 3460 3480 3500 3520 3540 3560 3580

Gauss

pH 4

pH 13

pH 7

King Midas of Phrygia – judging between Apollo and Pan

So, are natural phenolics antioxidants or prooxidants?

Financial support:

Romanian Ministry for Research and Innovation, grant

32PFE/19.10.2018 – CRESC-ITIM

National Science Centre, Poland, in a programme of

MAESTRO 6, application no. 2014/14/A/ST4/00640

Thank you!