Armacokinetic Analysis in the Compound Using the In Silico Technique The pharmacokinetic and physicochemical evaluation with the compound was performed utilizing the internet tool of SwissADME (http://www.swissadme.ch/), and gastrointestinal absorption, blood-brain barrier permeability, P-glycoprotein substrate, and cytochrome P450 inhibitory activities have been analyzed [36]. 3. Final results 3.1. Inhibitory Activities against the Enzymes and Antioxidant Activities with the Extracts Inhibitory activities against MAO-A, MAO-B, AChE, and BChE were mainly tested for 195 species of ELF extracts from Ukraine at 20 (MAO) or 50 /mL (ChE). The samples were screened based on the residual activities (Figures S1 4 in Supplementary Supplies). The cutoff values were 30 for MAO-B and 50 for MAO-A, AChE, and BChE. Hence, two samples for MAO-A, five for MAO-B, two for AChE, and 1 for BChE had been chosen (Table 1). ELF13 showed the lowest residual activity (19.7 ) for MAO-B; on the other hand, it exhibited no significant inhibitory activities for other enzymes. As a result, ELF13 was chosen for additional study and subjected for the cultivation, extraction, and isolation from the MAO-B inhibitor. ELF13 was identified as a fungus Daldinia fissa forming a symbiotic relationship with all the lichen Thamnolia vermicularis (Sw.) Schaer. Alternatively, antioxidant activity of 195 extracts of ELF was primarily measured at 100 /mL (Figure S5), and 3 extracts have been chosen according to the result from the DPPH antioxidant activity analysis (Table 2). ELF87 showed the highest inhibition (84.8 ), followed by ELF8 (58.5 ) at 100 /mL.J. Fungi 2021, 7,five ofTable 1. Inhibitory activities of monoamine oxidase-A (MAO-A), MAO-B, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) by endogenous lichen fungi (ELF) extracts. Residual Activity at 20 /mL ( ) ELF No. 13 22 26 68 71 73 74 110 172 MAO-A 26.2 5.76 43.6 2.47 MAO-B 19.7 5.67 22.2 six.87 26.2 1.40 28.0 1.32 27.0 1.16 AChE 41.0 three.83 40.eight 1.35 BChE 26.3 0.59 -Extracts were screened by a single assay of each and every enzyme inhibition, and after that efficient extracts had been moreover analyzed two occasions. The cutoff of residual activities were 30 for MAO-B and 50 for MAO-A, AChE, and BChE. The results are shown as mean and common deviation for α9β1 Gene ID triplicate experiments. -, not determined.Table two. DPPH (2,2-diphenyl-1-picrylhydrazyl) antioxidant activity of endogenous lichen fungi extracts. Inhibition at one hundred /mL ( ) ELF No. eight 84 87 DPPH 58.five 1.94 39.0 0.33 84.eight 0.inhibition = (absorbance of control–absorbance of reaction mixture)/absorbance of manage one hundred. The results are expressed as mean and standard deviation by duplicate experiments.three.2. Isolation of Compounds from Endogenous Lichen Fungi 13 (ELF13) Applying Prep Thin-Layer Chromatography (TLC) A total six L with the culture (200 mL 30 flasks) of ELF13 was extracted and concentrated to isolate compounds. Eight spots appeared around the PTLC plate together with the primary solvent and compounds within the spots had been recovered. The inhibitory activities against MAO-B by the compounds have been confirmed by way of the activity-guided method. Among the recovered eight fractions, fraction 1 showed the lowest residual activity (10.four ), and also other fractions showed greater residual activities of 50 (Figure 1). Inside the additional PTLC using the secondary solvent, two spots, C1 and C2, were identified and recovered to become 1 mg and 17 mg (96.eight , purity checked by high-performance liquid chromatography (HPLC)), Adenosine A2B receptor (A2BR) Antagonist MedChemExpress respectively, from 600 mg of fungal ext.