**3. Biological Activity**

#### *3.1. Assessment of Compounds and Extract for Free Radicals Scavenging and Antioxidant Activity*

The amphiphilic nature of the ABTS•<sup>+</sup> cation was used to identify both hydrophilic and hydrophobic antioxidants in dietary materials, whereas the DPPH- radical was used to measure an antioxidant's reducing power [30]. These fundamental chemical experiments reveal the radical scavenging and reduction characteristics of the potential antioxidant candidates.

Acetone extract (AE) and all isolated compounds (**1**–**14**) scavenged ABTS•<sup>+</sup> and DPPHradicals and the results are presented in Table 2. The results have demonstrated that AE and all the other compounds potently neutralized ABTS•<sup>+</sup> radicals (more than 70%) and have shown activity equal to the ascorbic acid standard, except for compound **13**, which only scavenged radicals by 50%.The pattern and potentials in decreasing order of ABTS•<sup>+</sup> scavenging potentials were observed as follows:olivetolic acid (**12**) > compound **5** > divarinolmonomethylether (**13**) > decarboxydivaricatic acid (**8**) > 4-*O*-methylnorhomo sekikaic acid (**2**) > atranol (**14**) > divaricatic acid (**7**) > decarboxystenosporic acid (**9**) > sekikaic acid (**1**) > 2,4-dimethyldivaric acid (**6**) > homo sekikaic acid (**3**) > methyldivaricatinate (**10**). In the case of the DPPH-radical scavenging assay, acetone extract (AE) and compound **14** scavenged DPPH-radicals potently by more than 50%, whereas **1**, **2**, **3**, **4**, **5**, **6**, **12**, and **13** countervailed DPPH-radicals (20–40%) moderately. It is important to mention that potent ABTS•<sup>+</sup> scavenging activities were observed in all compounds, but DPPH scavenging activity was detected to be moderate in all compounds except compound **14** (Table 2). As ABTS•<sup>+</sup> is a planar radical, it can be used to identify antioxidants even with low redox

potentials. However, due to the steric barrier of the N• radical, they may react slowly or not at all when tested on DPPH radicals [31]. This might be the reason why extracts and compounds scavenge ABTS•<sup>+</sup> more potently than DPPH-radicals. To check whether this radical scavenging activity is related to antioxidant properties, we challenged genomic DNA with hydrogen peroxide (H2O2)-induced oxidative damage.


**Table 2.** Free radical scavenging activities of AE and compounds (**1**–**14**) of *Ramalina conduplicans*.

ND = Not determined. The activity is expressed as % scavenging with regard to ascorbic acid scavenging activity. The SC50 is indicated for the most active compounds.
