Antioxidant and cytotoxic activities of quinones from Cetraria laevigata

Abstract Chemical investigation of the extracts obtained from the red thallus tips from Cetraria laevigata resulted in the isolation of five known quinoid pigments identified by FT-IR, UV, NMR, MS methods and by comparison with literature data (skyrin (1), 3-ethyl-2,7-dihydroxynaphthazarin (2), graciliformin (3), cuculoquinone (4) and islandoquinone (5)). An antioxidant capacity of compounds 1–5 were evaluated and compared with quercetin using a lipid peroxidation inhibitory assay and superoxide radical (SOR), nitric oxide radical (NOR), 1,1-diphenyl-2-picrylhydrazine (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) scavenging assays. Compounds 2, 4 and 5 were far more active: they demonstrated the antioxidant capacity in various test assays with the IC50 values 5–409 µM comparable to the flavonoid quercetin. While, the isolated quinones (1–5) exhibited weak cytotoxicity in human cancer cell line A549 assessed by MTT assay. GRAPHICAL ABSTRACT


Introduction
The species of Cetraria genus (Parmeliaceae) are common in the arctic, alpine, and boreal vegetation zones in the Northern Hemisphere, but few species also occur in cold areas of the Southern Hemisphere (Kärnefelt 1979).Most of the Cetraria species contain lichen substances such as protolichesterinic, lichesterinic, protocetraic, and fumarprotocetraic acids, physiochemical properties and biological activity of which had been studied earlier (Xu et al. 2016).In addition, to these compounds, some Cetraria species contain brightly colored pigments in the red thallus tips, composition, and properties of which remained unclear for a long time.

Results and discussion
Many of the natural quinoid pigments are known to show antioxidant activity.Polyhydroxylated naphthoquinone pigments of sea urchins exhibit antioxidant action and prevent the interaction of denatured proteins (Utkina and Pokhilo 2012).Skyrin in a wide concentration range (0.0001-100 µM) had only a slight ability to scavenge DPPH radicals, and the percentage of scavenged DPPH radicals in skyrin samples did not surpass the 5% level (Zajičková et al. 2022).
To evaluate the potential usefulness of compounds 1-5 we assessed the antioxidant activity of those using chemical assays to determine the inhibiting capacity of the lipid peroxidation (LP), the scavenging of the superoxide radical (SOR), nitric oxide radical (NOR), DPPH radical and ABTS •+ radical cation.The antioxidant activity of quinoid pigments 2-5 was studied for the first time.The positive control for all experiments was the flavonoid antioxidant quercetin.
It is well known that the number and arrangement of hydroxyl groups in polyphenols play a significant role in the antioxidant properties of compounds.The tested pigments have 4-6 hydroxyl groups in the molecules, but not all of these act as hydrogen donors to scavenge free radicals.Compounds 2, 4 and 5 have the highest antiradical activity including a DPPH and ABTS •+ radicals scavenging.The compounds 2, 4, 5 belong to naphthoquinones and are characterized by two OH groups in the ortho position.Probably, ortho relationships between OH groups could increase the radical scavenging ability of these compounds.
Quinones still comprise one of the largest classes of antitumor agents (Asche 2005).The cytotoxic effects of quinones are mainly exerted through DNA topoisomerase-II inhibition as well as superoxide radical formation when the quinone is reduced by reductases to a semiquinone radical (O'Brien 1991;Asche 2005).MTT assay was performed using HepG2 cells treated with 1 the most significant decrease in viability was observed in samples treated with 50-100 μM (Zajičková et al. 2022).While 1 did not exert cytotoxic effects on human lymphocytes.Bisanthraquinone rugulosin (diastereoisomer of 3) had cytotoxicity toward HL-60 cells with IC 50 = 9 μM (Ueno et al. 1995).
All of the compounds were examined for their cytotoxic effects against human lung cancer cell line A549 using the MTT assay at a concentration range of 12.5-100 μM.Only graciliformin (3) and islandoquinone (5) showed a weak inhibitory effect on the A549 cell growth (Figure S2) at high concentrations (50 and 100 μM).In fact, the viability was 63% and 67% in cells exposed to 100 μM of 3 and 5, respectively.Whereas compounds 1, 2, and 4 in our experimental conditions, revealed no cytotoxic effect against A549 cells.

Conclusion
Thus, the five quinoid pigments isolated from the red thallus tips of C. laevigata presented promising results at the antioxidant assays.Compounds 2, 4 and 5 were found to be the most active quinoid pigments and had an antioxidant activity comparable to the natural flavonoid quercetin.While all of the isolated quinones exhibited weak cytotoxicity in the studied concentration range toward A549 cell line.It is noteworthy that this is the first communication on the isolation of 1, 3 from the genus Cetraria and the antioxidant activity and cytotoxicity of compounds 2-5.