Biological Activities of Lichen-Derived Monoaromatic Compounds

Lichen-derived monoaromatic compounds are bioactive compounds, associated with various pharmacological properties: antioxidant, antifungal, antiviral, cytotoxicity, and enzyme inhibition. However, little is known about data regarding alpha-glucosidase inhibition and antimicrobial activity. Very few compounds were reported to have these activities. In this paper, a series of monoaromatic compounds from a lichen source were isolated and structurally elucidated. They are 3,5-dihydroxybenzoic acid (1), 3,5-dihydroxybenzoate methyl (2), 3,5-dihydroxy-4-methylbenzoic acid (3), 3,5-dihydroxy-4-methoxylbenzoic acid (4), 3-hydroxyorcinol (5), atranol (6), and methyl hematommate (7). To obtain more derivatives, available compounds from the previous reports such as methyl β-orsellinate (8), methyl orsellinate (9), and D-montagnetol (10) were selected for bromination. Electrophilic bromination was applied to 8–10 using NaBr/H2O2 reagents to yield products methyl 5-bromo-β-orsellinate (8a), methyl 3,5-dibromo-orsellinate (9a), 3-bromo-D-montagnetol (10a), and 3,5-dibromo-D-montagnetol (10b). Compounds were evaluated for alpha-glucosidase inhibition and antimicrobial activity against antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Compound 4 showed stronger alpha-glucosidase inhibition than others with an IC50 value of 24.0 µg/mL. Synthetic compound 9a exhibited remarkable activity against Staphylococcus aureus with a MIC value of 4 µg/mL. Molecular docking studies were performed to confirm the consistency between in vitro and in silico studies.

Lichen-derived monoaromatic compounds were divided into two sub-classes, orcinol, and β-orcinol, depending on the presence of a substituent at C-3. However, little is known about the antimicrobial and alpha-glucosidase inhibition of monoaromatic compounds and derivatives. As a few examples, orcinol, methyl orsellinate, ethyl orsellinate, and methyl β-orsellinate showed antimicrobial activities on various microorganisms with MIC values in the range of 30-500 µg/mL [6,14]. Regarding alpha-glucosidase inhibition, methyl hematommate [11] and cristiferide A [7] showed the potent activity while methyl orsellinate and methyl β-orsellinate are weak inhibitors [15,16]. Synthetic derivatives of monoaromatic compounds were produced mainly through esterification and etherification [15,16] but their alpha-glucosidase inhibition has not been studied.
In this study, seven natural monoaromatic compounds 1-7 were isolated from the lichen Parmotrema cristiferum (Figure 1). In addition, four synthetic compounds 8a, 9a, 10a, and 10b were prepared from the corresponding starting materials 8-10 ( Figure 2). Their structures were elucidated by spectroscopic data analysis and comparison with literature data. Compounds were evaluated for alpha-glucosidase inhibition and antimicrobial activity against antibiotic-resistant, pathogenic bacteria E. faecium, S. aureus, and A. baumannii.
In this study, seven natural monoaromatic compounds 1-7 were isolated from the lichen Parmotrema cristiferum (Figure 1). In addition, four synthetic compounds 8a, 9a, 10a, and 10b were prepared from the corresponding starting materials 8-10 ( Figure 2). Their structures were elucidated by spectroscopic data analysis and comparison with literature data. Compounds were evaluated for alpha-glucosidase inhibition and antimicrobial activity against antibiotic-resistant, pathogenic bacteria E. faecium, S. aureus, and A. baumannii.

Molecular Docking Studies
The pose views showed that 9a was strongly positioned in the 1t2p binding site by three h-bonds with Thr 180, Val 168, and Asn 114 and two halogen bonds with Asn 114 and Pro 163 (Figure 4), in which, the contribution to the binding site mechanism of residues such as Val 168, Thr 180, and Pro 163 has been demonstrated in the literature [21,22]. The free energy of this ligand was outstanding at −6.7 kcal/mol.   The in vitro alpha-glucosidase inhibitory activity of natural and synthetic compounds was evaluated (Table 1). Compounds 1-5, 7, 8a, 9a, 10a, and 10b displayed potent alphaglucosidase inhibitory activity with IC 50 values in the range of 24.0-171.1 µg/mL, compared with the 317 µg/mL of the acarbose positive control. Compounds 6 and 8-10 are inactive. Compounds 1-4 showed more potent activity than others, indicating the important role of 3,5-dihydroxy-1-carboxylbenzenoid ring in alpha-glucosidase inhibition. The 2,4dihydroxy-1-carboxylbenzyl moiety of compounds 8-10 might decrease the activity. The results were similar to those reported by Lopes and co-workers (2008) [16]. Compound 7 was stronger than 6, 8, and 9, proposing that the 3-CHO group exerted a significant effect on the activity. This was consistent with the data reported by Devi and co-workers (2021) [11]. Table 1. Alpha-glucosidase inhibitory activity of compounds 1-10, 8a, 9a, 10a, and 10b.

Molecular Docking Studies
The pose views showed that 9a was strongly positioned in the 1t2p binding site by three h-bonds with Thr 180, Val 168, and Asn 114 and two halogen bonds with Asn 114 and Pro 163 (Figure 4), in which, the contribution to the binding site mechanism of residues such as Val 168, Thr 180, and Pro 163 has been demonstrated in the literature [21,22]. The free energy of this ligand was outstanding at −6.7 kcal/mol.

Molecular Docking Studies
The pose views showed that 9a was strongly positioned in the 1t2p binding site by three h-bonds with Thr 180, Val 168, and Asn 114 and two halogen bonds with Asn 114 and Pro 163 (Figure 4), in which, the contribution to the binding site mechanism of residues such as Val 168, Thr 180, and Pro 163 has been demonstrated in the literature [21,22]. The free energy of this ligand was outstanding at −6.7 kcal/mol.     Generally, the estimated binding energies of the four ligands were consistent with the number of key interactions observed. However, with the central nucleus containing only one benzene nucleus and surrounded by polar functional groups (hydroxyl, ester, carboxylic acid), these four ligands lack the active support of hydrophobic interactions, which may explain the low affinity of those ligands to the target protein. The free energies and major residues of those complexes are presented in Table 3.  Generally, the estimated binding energies of the four ligands were consistent with the number of key interactions observed. However, with the central nucleus containing only one benzene nucleus and surrounded by polar functional groups (hydroxyl, ester, carboxylic acid), these four ligands lack the active support of hydrophobic interactions, which may explain the low affinity of those ligands to the target protein. The free energies and major residues of those complexes are presented in Table 3.

Source of the Lichen Material P. cristiferum
The thallus of lichen P. cristiferum was collected in Duc Trong district, Lam Dong province, Vietnam, in March 2020. The scientific name of the lichen was determined by Dr. Thi-Phi-Giao Vo, Faculty of Biology, Ho Chi Minh University of Science, National University-Ho Chi Minh City. A voucher specimen (UE-L006) was stored in the herbarium of the Department of Organic Chemistry, Ho Chi Minh University of Education.

General Procedure to Synthesize Compounds 8a and 9a
In 2.0 mL of mixture of acetic acid and DMSO (3:1, v/v), methyl β-orsellinate (8,10.0 mg, 0.051 mmol), and sodium bromide (15.76 mg, 0.153 mmol) were dissolved at 80 • C under stirring. Then, 0.5 mL of 30% hydrogen peroxide (4.847 mmol) was added to the reaction flask. The reaction was conducted for 30 min and was periodically monitored every 5 min by TLC. After the reaction mixture was neutralized with saturated sodium hydrogen carbonate, it was further extracted with ethyl acetate-water (1:1, v/v) to gain an organic layer. The organic layer was washed thoroughly with brine three times, then dried and applied to silica gel column chromatography (CC), eluted with n-hexane-EtOAc-acetone (10:1:2, v/v/v) to obtain 8a (13.3 mg, 95%).

General Procedure to Synthesize Compounds 10a and 10b
In 5.0 mL of mixture of acetic acid, D-montagnetol (10, 50.0 mg, 0.184 mmol) and sodium bromide (56.9 mg, 0.552 mmol) were dissolved at room temperature. Then, 1.8 mL of 30% hydrogen peroxide (17.487 mmol) was added to the reaction mixture. The reaction was conducted for 30 min. The resulting solution was neutralized with saturated sodium hydrogen carbonate, then extracted with ethyl acetate-water (1:1, v/v) to gain an organic layer. This layer was subsequently washed with brine three times, then dried and applied to silica gel CC, eluted with n-hexane-EtOAc-acetone-water (2:2:2:0.01, v/v/v) to obtain 10a (24.5 mg, 38%) and 10b (41.6 mg, 53%).

Antimicrobial Activity Assay
The agar well diffusion method was utilized to investigate the antibacterial activity of the isolated compounds on antibiotic-resistant, pathogenic bacteria E. faecium, S. aureus, and A. baumannii. Three bacterial pathogens were cultured in nutrient broth at 37 • C for 18 h. The cultures were diluted with sterile 0.9% NaCl to obtain bacterial solutions of 1.5 × 10 8 CFU/mL. This solution with a volume of 100 µL was spread on a Mueller-Hinton agar plate. Holes with a diameter of 8 mm were punched aseptically to create wells on the surface of the Mueller-Hinton agar. The compounds were dissolved in DMSO. The amount of 50 µg of each compound solution was inserted into the wells. The plates were incubated at 37 • C for 16-18 h and the antibacterial activity of each compound was recorded by measuring the diameters of the inhibition zones surrounding the wells. The determination of the minimum inhibitory concentration of 9a against Staphylococcus aureus was performed by the agar dilution method [23]. Compound 9a was dissolved in DMSO to the final concentration of 1 mg/mL and then diluted with Mueller-Hinton agar (MHA) to the concentration range of 0, 1, 2, 4, 8, 16, 32, 64 µg/mL as the final concentrations in MHA plates. S. aureus was cultured in nutrient broth at 37 • C overnight with shaking. The bacterial culture was diluted with sterile 0.9% NaCl to the concentration of 10 7 CFU/mL. 1 µL of the diluted bacterial solution (10 4 CFU) was placed on the surface of the MHA plates and the plates were incubated at 37 • C for 16-18 h. The MIC value was recorded as the lowest concentration of 9a that inhibited the growth of S. aureus. Kanamycin was chosen as the positive control in this experiment, while DMSO was regarded as a control.

Molecular Docking Studies
The PDB structures of proteins (4j5t and 1t2p) were downloaded from the Protein Data Bank, while the 3D structures of ligands were modeled via the website chemicalize.com. After the conversion from PDB files into a PDBQT format by AutodockTools, the docking Institutional Review Board Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in Supplementary Material.