*3.3. Assessment of In Vitro Antihyperglycemic Activity of Compounds and Extract as Intestinal α-Glucosidase Enzyme Inhibition*

The α-glucosidase enzyme is a key enzyme that catalyses disaccharide digestion. The inhibition of α-glucosidase in the intestine slows digestion and the overall rate of glucose absorption into the blood. This has proven to be one of the most effective ways for lowering post-prandial blood glucose levels and, as a result, preventing the onset of late diabetes complications [33]. Sekikaic acid (**1**) was already recognized to inhibit α-glucosidase along with usnic acid and salazinic acid from other *Ramalina* species, but it is not the most effective compound [34]. As per Figure 6, it was stated that acetone extract (AE) and compound **4** have displayed better α-glucosidase inhibition (*p* < 0.01) than Acarbose.

**Figure 5.** In vitro DNA damage assay. Compounds (**1**–**14**) and *R. conduplicans.* AE were incubated with DNA and Fenton's Reagent and DNA damage was recorded with Agarose Gel electrophoresis. Respective graphical representation. ### *p* < 0.001; vs. control (DMSO + DNA). \*\*\* *p* < 0.001, \* *p* < 0.05; vs.DMSO + DNA + FR, One-way ANOVA followed by Tukey's multiple comparison test was used to calculate values. Values are represented as mean ± SD, *n* = 3. AE = Acetone Extract, FR = Fenton's Reagent.

**Figure 6.** Intestinal α-glucosidase inhibitory assay. Compounds (**1**–**8**), (**11**–**14**), and acetone extract (AE) were incubated with α-glucosidase enzyme and the release of *p*-nitrophenol was determined. \*\*\* *p* < 0.001, \*\* *p* < 0.01; vs. Acarbose. One-way ANOVA followed by Tukey's multiple comparison test was applied to compare differences. Values are represented as mean ± SD, *n* = 3. AE = Acetone Extract. Activity was not detected for compounds **9** and **10**.

On the other hand, compounds **8**, **11**, **12**, and **14** demonstrated inhibitory effects comparable to those of the standard Acarbose (Figure 6). As contrasted activities can be observed between structurally-related compounds, structure–activity relationships can be considered. This is the case between depsides **3** and **4,** suggesting a positive influence of the C1-pentyl chain with regard to substitution by a C1-propyl chain. When this length modification of the alkyl chain occurs on the B ring of decarboxylated derivatives (active compound **8** versus inactive compound **9**) the opposite influence can be observed. The presence of a C6 -carboxylic group lowers the α-glucosidase inhibitory activity as compound **7** is less active than compound **8**. Methylation of the carboxylic function of the monoaromatic divaricatinic acid **11** resulted in a complete loss of activity. Nevertheless, most of the tested compounds were found with some activity, such as compounds **1**, **2**, **3**, **5**, **6**, **7**, and **13,** which displayed mild to moderate enzyme inhibition (*p* < 0.001). These results are to be pooled with the growing number of reports on the antidiabetic potential of lichen extracts or molecules [34–36]. The combination of activities with different mechanisms of action is of particular interest to develop potent antihyperglycemic effects. Lowering glucose absorption and limiting oxidative damages due to hyperglycemia, as expected from the lichen extract, could be promising. The challenge is to use standardized extracts that were previously checked to be safe for acute and chronic intake.
