Phenolic Secondary Metabolites and Antiradical and Antibacterial Activities of Different Extracts of Usnea barbata (L.) Weber ex F.H.Wigg from Călimani Mountains, Romania

Phenolic compounds represent an essential bioactive metabolites group with numerous pharmaceutical applications. Our study aims to identify and quantify phenolic constituents of various liquid and dry extracts of Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) from Calimani Mountains, Romania, and investigate their bioactivities. The extracts in acetone, 96% ethanol, and water with the same dried lichen/solvent ratio (w/v) were obtained through two conventional techniques: maceration (mUBA, mUBE, and mUBW) and Soxhlet extraction (dUBA, dUBE, and dUBW). High-performance liquid chromatography with diode-array detection (HPLC-DAD) was performed for usnic acid (UA) and different polyphenols quantification. Then, the total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity (AA) were determined through spectrophotometric methods. Using the disc diffusion method (DDM), the antibacterial activity was evaluated against Gram-positive and Gram-negative bacteria known for their pathogenicity: Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 13883). All extracts contain phenolic compounds expressed as TPC values. Five lichen extracts display various UA contents; this significant metabolite was not detected in dUBW. Six polyphenols from the standards mixture were quantified only in ethanol and water extracts; mUBE has all individual polyphenols, while dUBE shows only two. Three polyphenols were detected in mUBW, but none was found in dUBW. All U. barbata extracts had antiradical activity; however, only ethanol and acetone extracts proved inhibitory activity against P. aeruginosa, S. pneumoniae, and S. aureus. In contrast, K. pneumoniae was strongly resistant (IZD = 0). Data analysis evidenced a high positive correlation between the phenolic constituents and bioactivities of each U. barbata extract. Associating these extracts’ properties with both conventional techniques used for their preparation revealed the extraction conditions’ significant influence on lichen extracts metabolites profiling, with a powerful impact on their pharmacological potential.

With numerous pharmacological activities, the lichens of the genus Usnea (Parmeliaceae) are appreciated as powerful phytomedicines, used for therapeutical purposes for thousands of years [18]. The most known secondary metabolite in Usnea sp. is usnic acid-a phenolic compound with a dibenzofuran structure. As yellow crystals, it is found on cortex fungal hyphae, exhibiting a photoprotective action [19]. Usnic acid is found as a (+) enantiomer in Usnea lichens [20]. A valuable representative of this genus, known for its antioxidant [21], antibacterial [22], and photoprotective [7] effects, is U. barbata. Usnic acid is the main secondary metabolite responsible for its pharmacological potential [23]. The pharmaceutical applications of UA as an antibacterial agent are limited by its poor water solubility [24] and significant hepatotoxicity [25]. Therefore, the nanosystems with usnic acid must be able to increase its bio-disponibility, tolerance, and antibacterial effects [26]. Interesting nanoformulations were performed: liposomal UA-cyclodextrin inclusion complexes, which increase usnic acid solubility in water [27], glycosylated cationic liposomes, promoting usnic acid penetration in the bacterial biofilm matrix [28], and magnetic nanoparticles [29] with antimicrobial activity and antibiofilm activity against Gram-positive bacteria (S. aureus and E. faecalis) and Gram-negative ones (P. aeruginosa). Balaz et al. [30] recently proposed a bio-mechanochemical synthesis of silver nanoparticles using U. antarctica and other lichen species. Using AgNO 3 (as a silver precursor) and lichens (as reduction agents), they performed techniques of mechanochemistry (ball milling) and obtained nanoparticles with an intense antibacterial effect against S. aureus. This described procedure overcomes the lichen secondary metabolites' low solubility in water. Siddiqi et al. [31] demonstrated the antimicrobial properties of U. longissima-driven silver nanoparticles through the denaturation of ribosomes, leading to enzyme inactivation and protein denaturation, resulting in bacterial apoptosis.
Numerous studies investigated the antibacterial effects of Usnea sp. Extracts-obtained through conventional and green extraction techniques-for pharmaceutical applications [39]. Thus, Tosun et al. [40] explored the antimycobacterial action of U. barbata fractions in petroleum ether, chloroform, methanol, and water. Bate et al. [41] studied the antibac- terial activity of U. articulata and U. florida methanol macerates against MDR bacteria (Staphylococcus sp., P aeruginosa, Salmonella sp., and E. coli). Zizovic et al. [42] proved the strong antibacterial action of U. barbata supercritical fluid extracts (SFE). One year later, Ivanovic et al. [43] analyzed the influence of various extraction conditions (temperature, pressure) and pre-treatment methods on bactericidal effects against S. aureus strains. Basiouni et al. [44] evaluated the U. barbata sunflower oil extract inhibitory activity on bacterial strains isolates from poultry. In a previous study, Matvieva et al. [15] analyzed the antimicrobial properties of the ethanol, isopropanol, acetone, DMSO, and water extracts of Usnea sp against S. aureus, B. subtilis, and E coli.
We propose to investigate the antibacterial and antiradical properties of U barbata extracts in the same solvents, obtained by two low-cost and easy-to-use conventional techniques. Our study novelty consists of a comparative analysis of fluid and dry U. barbata extracts in ethanol, acetone, and water, obtained by maceration and Soxhlet extraction [34], determining their phenolic constituents and evaluating the free radical scavenging activity and antibacterial effects. Our results revealed that, despite the same ratio between the dried lichen and the solvent (w/v), all U. barbata extracts display significant differences in the phenolic metabolites' diversity and amount due to extraction conditions, with a substantial impact on their bioactivities.

Lichen Extracts
All data regarding the obtained U. barbata extracts are displayed in Table 1 and Figure S1 from Supplementary Materials. Data from Table 1 show that the extraction temperature for liquid extracts was 20-22 • C, and their color varies from yellow (mUBA) to light brown (mUBE) and brownreddish (mUBW).

HPLC-DAD Determination of Usnic Acid Content
The usnic acid contents in all U. barbata extracts are displayed in Table 2. All liquid extracts contain UA. Thus, mUBA had the highest UA content (211.9 mg/g extract equivalent to 21.19 mg/g dried lichen), following in decreasing order mUBE (0.257 mg/g, corresponding to 0.025 mg/g dried lichen) and mUBW (0.045 mg/g corresponding to 0.004 mg/g dried lichen). According to https://pubchem.ncbi.nlm.nih. gov/compound/Usnic-acid (accessed on 20 May 2022), usnic acid solubility significantly decreases in order: acetone > ethanol > water; these data can explain our results. The chromatograms of usnic acid standard and U. barbata extracts in all three solvents are displayed in Figure 1. Data from Table 2 show that only two dry extracts contain UA because in dUBW it was non-detected. Dry acetone extract contains UA of 241.773 mg/g, corresponding to 13.418 mg/g dried lichen. The usnic acid content in dUBE is 108.752 mg/g (12.125 mg/g dried lichen).

HPLC-DAD Determination of Polyphenols
The polyphenols contents are displayed in Table 3     As can be seen, six polyphenols of the standard mixture were identified only in ethanol and water fluid extracts; their high solubility in polar solvents could justify their absence in acetone extracts (Table 3 and Figures 2 and 3).    As can be seen, six polyphenols of the standard mixture were identified only in ethanol and water fluid extracts; their high solubility in polar solvents could justify their absence in acetone extracts (Table 3 and Figures 2 and 3).  As can be seen, six polyphenols of the standard mixture were identified only in ethanol and water fluid extracts; their high solubility in polar solvents could justify their absence in acetone extracts (Table 3 and Figures 2 and 3). Of all six polyphenols identified in mUBE: caffeic acid (CA), p-coumaric acid (pCA), ellagic acid (EA), chlorogenic acid (ChA), gallic acid (GA), and cinnamic acid (CiA), only two (EA and GA) were found in dUBE, and three (pCA, ChA, and GA) in mUBW (Figures 2-4). The common polyphenol for all three extracts is GA, with the highest content in mUBW (60.358 mg/g), followed by mUBE (27.487 mg/g) and dUBE (0.870 mg/g). Ellagic acid content is 230.819 mg/g in mUBE and 0.605 mg/g in dUBE (Table 3, Figure 4).
The common polyphenols for mUBW and mUBE were pCA and ChA; their amounts were higher in mUBW (0.749 and 0.627 mg/g) than mUBE (0.312 and 0.512 mg/g). The other two polyphenols-CA (0.414 mg/g) and CiA (17.948 mg/g)-were identified exclusively in mUBE (Table 3, Figure 2).
The dUBW chromatogram ( Figure 5) shows three peaks at the following retention times (RT): 15.113 min, 15.642 min, and 16.091 min; these RT values differed from standard polyphenols' ones. Their absence in dUBW could be due to their thermolability; the Soxhlet extraction involves prolonged heating for 8 h at 95-100 • C [45].
The polyphenols from the standard mixture were also non-detected in both U. barbata acetone extracts (Table 3) because their solubility is lower in this solvent than in ethanol or water.

Total Phenolic Content
It can be observed that the highest total phenolic content (TPC) values belong to dry U. barbata extracts ( Table 4). The dUBA had the highest TPC (862.843 mg PyE/g); it is followed in decreasing order by dUBE (573.234 mg PyE/g) and dUBW (111.626 mg PyE/g). The TPC values in fluid extracts decreased in the following order: mUBE (276.603 mg PyE/mL), mUBA (220.597 mg PyE/mL), and mUBW (176.129 mg PyE/mL). TPC includes usnic acid, identified polyphenols, and unidentified phenolic constituents of each U. barbata extract.

Free-Radical Scavenging Activity Assay
The results are displayed in Table 4. Data from Table 4 show that all U. barbata extracts have antiradical activity. This effect was higher for dry ethanol and acetone extracts (16.728% for dUBE, 15.471% for dUBA) than fluid ones (12.162% for mUBE, 11.146% for mUBA). Only for water extracts, the antiradical activity of dUBW (3.951%) is lower than the mUBW one (6.429%).

Antibacterial Activity
The obtained results proved that the negative control (DMSO 0.1%) has no inhibitory effect on the bacteria tested (IZD = 0 mm). Only U. barbata extracts in acetone and ethanol inhibited bacterial strains' growth. ( Figure S2, Supplementary Materials). Neither UBWs have any inhibitory effect on the tested bacteria (IZD = 0 mm).
Given that usnic acid is the major secondary metabolite of the genus Usnea, we considered this phenolic compound as a positive control. For the optimal interpretation of the obtained IZD values, we used two bactericidal antibiotics with different mechanisms of action and breakpoints: ofloxacin (OFL) and ceftriaxone (CTR). The data displayed in Table 5 show the IZD values (mm) for all U. barbata extracts, UA, and standard antibiotics drugs (OFL and CTR). Standard antibacterial drugs breakpoints * Ofloxacin Therefore, comparing the IZD values of the U. barbata extracts to those of both standard antibiotics on S. aureus, none had antibacterial action (IZD = 11.00-13.66 mm). Only usnic acid has an IZD (16.33 mm) in the "I" range of ofloxacin (17-15 mm); this means that antibacterial activity on S. aureus is dose dependent. Compared to ceftriaxone, the IZD value for UA belongs to the resistance range (<20 mm).
S. pneumoniae is sensitive to all U. barbata extracts as well as to usnic acid (IZD = 17.33-18.67 mm) when IZD values are compared to ofloxacin (S ≥ 16 mm *). However, it could be considered resistant when IZD values were compared to CTR (S ≥ 26 mm *).
Considering the data registered in Table 5, we calculated the antibacterial activity index (AI), reporting the IZD values (mm) of lichen extracts to the ones of the standard antibiotic drugs [47]. It can be noted that dry and fluid U. barbata acetone and ethanol extracts had similar inhibitory effects (Table 6). The presence of similar bioactive secondary metabolites, the fluid extracts used after solvent evaporation, and the additional presence of the polyphenols known for their strong antibacterial action could explain the results registered in Tables 5 and 6. Thus, UA had the highest inhibitory activity on S. aureus, showing a dose-dependent antibacterial effect and the highest AI values; the following are the extracts with a high usnic acid content, respectively UBA. U. barbata ethanol extracts show the lowest inhibitory effect because usnic acid is known for its highest inhibition levels on S. aureus; both UBEs have lower UAC values than the corresponding UBAs ones (Table 5).
On S. pneumoniae and P. aeruginosa, the lichen extracts in ethanol indicated the most significant inhibitory levels. Antibacterial activities of individual polyphenols could justify these results. They showed an antibacterial action against S. pneumoniae similar to ofloxacin. On S. pneumoniae, the AI values compared to OFL are statistically different from those linked to CTR (Table 6). In this case, for all U. barbata extracts, AI ≥ 0.912, proving that their antibacterial activity is similar to OFL. Against P. aeruginosa, mUBA and dUBA reported AI values higher than OFL (AI > 1) and similar to CTR (AI ≥ 0.952) ( Table 6).

Data Analysis
We obtained U. barbata extracts performing two easy-to-use and low-cost conventional techniques mentioned in Romanian Pharmacopoeia X [48]: maceration for fluid extracts and Soxhlet extraction for dry ones. They have been one of the most used extraction procedures for herbal bioactive compounds [49]. According to the green chemistry concept, the solvents used for lichen extraction are "preferable," having low toxicity and significant safety [50]. Our entire study's data were synthesized in Table 7.
From the beginning, the same ratio-1:10 (w/v) between dried lichen and solventwas maintained for all extracts. The fluid extracts were obtained at room temperature (20-22 • C). The Soxhlet extraction was performed by prolonged heating, and the requested temperature values registered in Table 7 were maintained for 8 h.
The phenolic metabolites contents were strongly influenced by extraction conditions, as shown in Table 7. Usnic acid content and TPC significantly increase in acetone and ethanol dry extracts than in fluid ones; UBAs have higher UAC and TPC than UBEs. The mUBW had the lowest TPC and UAC. However, after 8 h of Soxhlet extraction at 100 • C, dUBW shows diminished TPC values and no UAC.
The individual polyphenols were quantified only in ethanol and water U. barbata extracts. The mUBE contains all six polyphenols (CA, CiA, pCA, EA, GA, and ChA) and mUBW-only three (pCA, GA, and ChA). Regarding the corresponding dry extracts, in dUBE only two polyphenols (EA and GA) were found in lower content than mUBE; dUBW has no polyphenols. These detailed aspects could be explained in the first step by the solubility differences of phenolic compounds in each extraction solvent. Polyphenols are soluble in polar solvents (ethanol, water); however, they are affected by prolonged heating [45]; thus, it can justify their decreasing or absence in the dry extracts after Soxhlet extraction for 8 h at 75-80 • C (dUBE) and 95-100 • C (dUBA). The lowest solubility of usnic acid in water underlies the minimal UAC value in mUBW. The high temperature of extraction (100 • C for 8 h) affects usnic acid stability; thus, the absence of UA in dUBW could be justified. According to https://www.biocrick.com/Usnic-acid-BCN4306.html (accessed on 2 May 2022), usnic acid storage requests desiccation and freezing (−20 • C); this information supports our results.
On the other hand, it can be seen that the dry extracts are obtained with a considerably low yield. When all UAC values are reported to the dried lichen amount used for each extract preparation, 2.119% corresponds to mUBA and only 1.341% for dUBA.

Principal Component Analysis
Principal component analysis (PCA) was performed for all U. barbata liquid and dry extracts and variable parameters-according to the correlation matrix from Supplementary Materials-and illustrated in Figure 6. The lichen extracts' phytoconstituents significantly influence their pharmacological potential. Hence, we explored the metabolites content to explain the differences in the obtained results regarding antiradical and antibacterial effects. Then, we determined the correlations between these bioactivities and phenolic metabolites quantified in each lichen extract. All data are displayed in Figures 7-10 and detailed in Supplementary Materials. temperature (detailed data in Supplementary Materials). Usnic acid with temperature registered the lowest negative correlation (r = −0.042).
The lichen extracts' phytoconstituents significantly influence their pharmacological potential. Hence, we explored the metabolites content to explain the differences in the obtained results regarding antiradical and antibacterial effects. Then, we determined the correlations between these bioactivities and phenolic metabolites quantified in each lichen extract. All data are displayed in Figures 7-10      In mUBE, all quantified phenolic secondary metabolites significantly correlate with DPPH free radical scavenging ability (AA, r ≥ 0.930) and antibacterial activities ( Figure 7).
As expected, Figure 7   In mUBE, all quantified phenolic secondary metabolites significantly correlate with DPPH free radical scavenging ability (AA, r ≥ 0.930) and antibacterial activities ( Figure 7).
Data illustrated in Figure 8 highlight the strongest correlation (r = 0.999, p < 0.05) between phenolic compounds (EA, GA, and TPC) and AA and P.a. On P. aeruginosa, the powerful action of ellagic acid and gallic acid is due to phenolic compound general mechanisms and biofilm inhibition [52]. The same correlation (r = 0.999, p < 0.05) can be noticed between UA and S.a.; UA is a valuable antibacterial compound against S. aureus and, as a positive control, had a dose-dependent antibacterial effect. Both activities-AA and P.a.-are also highly correlated (r = 0.999, p < 0.05).
These correlations associated with the bio-activities of all quantified metabolites could explain the similar inhibitory activity on bacterial strains growing of both U barbata extracts in ethanol and acetone. Moreover, in these extracts, all phenolic metabolites could synergistically act.
The PCA-correlation circle for UBWs is displayed in Figure 10. Data from Figure 10 show that usnic acid (r = 0.910, p > 0.05) and individual polyphenols-pCA (r = 0.951, p > 0.05), GA and ChA (r = 0.999, p < 0.05) highly correlate with AA in liquid water extract. Furthermore, in both UBWs, TPC show a powerful correlation with AA (r = 0.995, and 0.961, p < 0.05). However, because the phenolic compounds with known antibacterial action were extracted in water in minimal quantities, both UBWs did not exhibit any inhibitory effect on bacteria tested (IZD = 0).
Our study deeply analyzed six U. barbata extracts in three solvents, from the description of extraction conditions to phenolic constituents' determination and the evaluation of their biological activities. A detailed data analysis was performed on the correlations between phenolic metabolites and biological activities for each U. barbata extract, aiming to explain the obtained results. We correlated phenolic metabolites with antiradical and antibacterial activities and with extraction temperature for all six U. barbata extracts. The extraction temperature's significant role was highlighted by comparing the liquid and dry extracts in the same solvent. Thus, we evidenced the strong influence of the extraction temperature on phenolic metabolites diversity and content and, consequently, the strong impact on antiradical and antibacterial activities.
Correlating and interpreting all data, we made each lichen extract characterization, highlighting the similar and different properties compared to the others (Figure 11).  Figure 11a shows that the fluid UBE (obtained at room temperature) contains UA in a low content and all six polyphenols in an appreciable amount. It can be noticed that CA, EA, and CiA are associated exclusively with mUBE; moreover, it shares ChA, GA, and pCA with mUBW. Individual polyphenols contribute considerably to the mUBE's TPC value (Figure 11a). These constituents could synergistically act, leading to their significant antiradical and antibacterial potential (Figure 11b). The Soxhlet extraction at 75-80 • C significantly diminished the polyphenols content; thus, dUBE reported low concentrations of only two polyphenols (EA and GA, Figure 11a). Moreover, UA and other phenolic secondary metabolites were resistant to prolonged heating and detected in dry acetone extract (Figure 11a). Therefore, dUBE shows a higher AA than mUBE and similar antibacterial effects. The fluid water extract (mUBW) shows the lowest content of phenolic metabolites compared to other macerates. It contains three individual polyphenols (pCA, GA, ChA) and usnic acid (Figure 11a). Despite the antibacterial properties of all phenolic constituents, their content is too low, and mUBW does not inhibit bacterial strains' growth; it has only moderate antiradical activity (Figure 11b). The prolonged heating at 100 • C during Soxhlet extraction diminished phenolics content; UA and individual polyphenols from mUBW were not detected in dUBW (Figure 11a), and AA decreased.
Both acetone extracts (mUBA and dUBA) have the same metabolites (UA and TPC) and bioactivities (Figure 11b); the temperature and yield have a quantitative influence, increasing UAC and TPC in dUBA. Therefore, AA augments and antibacterial properties are similar. In Figure 11a,b, both UBAs and dUBE are positioned at low distances; both UBWs are located in the same quarter of the PCA-biplot, thus evidencing their similar properties.

Discussion
The low yields associated with diminished UAC in dried lichen can also be observed in other studies on U. barbata extracts obtained in various conditions [42,43,53,54]. The most relevant data are displayed in Table 8. UB SFE-U. barbata extract obtained by supercritical fluid extraction with CO 2 , UBDEA-U. barbata dry extract in ethyl acetate, UBDA-U. barbata dry extract in acetone, UBDE-U. barbata dry extract in ethanol, UBDM-U. barbata dry extract in methanol, UBO-U. barbata extract in canola oil, RM-roller mill; UM-ultra-centrifugal mill; CM-cutting mill; RGD-rapid gas decompression. * Data registered in decreasing order; superscript letters evidenced the data adapted from: a [53], b [42], c [54], d [43].
The data from Table 8 indicate that the UAC (%) in dried lichen generally decreases directly proportional to the extraction yield when the same solvent is used.
The usnic acid chemical structure strongly relates to U. barbata antiradical and antibacterial activities [22]. Due to protonophore and uncoupling action, all three phenolic OH groups of UA are essential [55], leading to bacterial membrane potential dissipation, associated with bacterial colonies growing inhibition. Maciag-Dorszynska et al. [56] proved that usnic acid produces a rapid and strong inhibition of nucleic acids synthesis in Grampositive bacteria (S. aureus and B. subtilis). It could also inhibit Group A Streptococcus (Streptococcus pyogenes) biofilm formation [57], reducing biofilm biomass and depleting the biofilm-forming cells' proteins and fatty acids. Sinha et al. [58] proved that UA could act synergistically with norfloxacin and modify S. aureus methicillin-resistant (MRSA) drug resistance. This effect involves efflux pump inhibition, oxidative stress induction, and down-regulation of peptidoglycans and fatty acids biosynthesis. These mechanisms alter membrane potential and perturb cell respiration and metabolic activity.
The polyphenols could synergistically act with usnic acid and other secondary metabolites in U. barbata extracts' antiradical and antibacterial activities. The antibacterial effects of polyphenols implicate various mechanisms. Thus, Lou et al. [59] proved that the p-coumaric acid bactericidal effect against S. aureus and S. pneumoniae involves irreversible permeability changes in bacterial cell walls and binding to bacterial genomic DNA; as a result, it occurs cell function inhibition followed by bacteria cell death. Caffeic acid (CA) acts as an antibacterial drug through various mechanisms; it produces cell membrane depolarization and disruption, reduces the respiratory activity of bacteria, decreases efflux activity, affects intracellular redox processes, donates protons, and increases intracellular acidity [34]. Moreover, CA proved to have an appreciable inhibitory effect against S. aureus (IZD = 12 mm) [34]. Cinnamic acid (CiA) preferentially acts against Gram-negative bacteria (P. aeruginosa), determining cell membrane damage, affecting its lipidic profile, and leading to protein loss and denaturation [60]. Chlorogenic acid (ChA) antibacterial mechanisms involve outer cell membrane bounding and disrupting, intracellular potential exhausting, and loss of cytoplasm macromolecules, leading to cell death [61]. On S. pneumoniae, ChA inhibits a key virulence factor (neuraminidase) [62]. Gallic acid (GA) has a significant antibacterial effect against Gram-positive bacteria (S. aureus, Streptococcus sp.), increasing their ability to accept electrons. On Gram-negative bacteria, this property could decrease, indicating that GA is an electrophilic compound interacting with bacterial surface components [63][64][65]. Ellagic acid (EA) acts on S. aureus damaging the bacteria cell membrane, leading to significant leakage of proteins and nucleic acids. Its antibacterial activity could inhibit protein synthesis, inducing great morphological changes in bacterial cell structure [66]. Both phenolic acids (GA and EA) also proved bactericidal effects against P. aeruginosa [52]. In encapsulated form, their antibacterial potential could increase [38].
Numerous researchers analyzed the antibacterial activity of U. barbata and Usnea sp.; generally, their results were similar to those obtained in our study [39]. The sensibility of Gram-positive bacteria to usnic acid and various Usnea sp. extracts is most known. Idamokoro et al. [67] analyzed the effect of U. barbata extracts in methanol and ethylacetate against 13 isolated Staphylococcus sp. involved in cow mastitis. They evidenced ethyl-acetate extract's lower inhibitory activity than methanol ones. On S. aureus, they reported an IZD value = 14 mm for methanol extract, similar to our dUBA (IZD = 13.66 mm). Mesta et al. [68] indicated the IZD values of 12 mm-for U. ghatensis ethanol extract 15 mg/mL against S. aureus-and 18 mm-for U. undullata ethanol extract 15 mg/mL on S. pneumoniae; both values are similar to those for m/dUBE obtained in the present study. In a previous study [69], we evaluated the antibacterial activity of U. barbata liquid extracts against two other Streptococcus sp. (S. oralis and S. intermedius) isolated from the oral cavity. Those obtained IZD values proved that mUBE had a stronger action for both Streptococcus sp. than mUBA; mUBW did not show any inhibitory effect. No inhibitory effects (IZD = 0) displayed the extracts of U. pectinata, U. coraline, and U. baileyi in methanol and dichloromethane against K. pneumoniae [5]. The methanol extracts of U. articulata (IZD = 28 mm) and U. florida (IZD = 18 mm) highlighted a remarkable antibacterial action against P. aeruginosa [41]. U. florida extract in methanol also proved significant activity on S. aureus (IZD = 30). Boisova et al. [70] optimized the conditions of UA SFE extraction from U. subfloridana (for 80 min, at a temperature of 85 • C and pressure of 150 atm). Their obtained extract proved an intense antibacterial activity against S. aureus.
The bacterial lines were obtained from Microbiology Department, S.C. Synevo Romania SRL, Constanta Laboratory, in partnership agreement No 1060/25.01.2018 with the Faculty of Pharmacy, Ovidius University of Constanta. Culture media Mueller-Hinton agar simple and one with 5% defibrinated sheep blood were supplied by Thermo Fisher Scientific, GmbH, Dreieich, Germany.

Lichen Extracts
U. barbata was harvested from Călimani Mountains, Romania (47 • 28 N, 25 • 10 E, 900 m altitude) in March 2021. The lichen was dried at a constant temperature below 25 • C in an airy room, protected from the sunlight. After drying, the obtained herbal product was preserved for a long time in the same conditions for use in subsequent studies. The lichen was identified using standard methods by the Department of Pharmaceutical Botany of the Faculty of Pharmacy, Ovidius University of Constanta. A voucher specimen (Popovici 3/2021 Ph/UOC) [71] can be found at the Department of Pharmacognosy, Faculty of Pharmacy, Ovidius University of Constanta.
The dried lichen was ground in an LM 120 laboratory mill (PerkinElmer, Waltham, MA, USA) and passed through the no. 5 sieve [19]. The obtained moderately fine lichen powder (particle size ≤ 315 µm) was subjected to extraction in acetone, 96% ethanol, and water (dried lichen: solvent ratio (w/v) = 1:10) using two conventional techniques. The first procedure was maceration-three samples of 10 g ground dried lichen were extracted with 100 mL solvent (water, acetone, and 96% ethanol) in a dark place at room temperature (20-22 • C) for 10 days, with manual shaking 3-4 times/day. The resulting extractive solutions were filtered and made up of a 100 mL volumetric flask with each solvent. These fluid extracts (mUBA, mUBE, and mUBW) were preserved in dark-glass recipients with sealed plugs in the same conditions until processing.
The second one was Soxhlet extraction for 8 h, with the temperature values around each solvent's boiling point. Thus, three samples of 20 g ground dried lichen were refluxed at Soxhlet for eight hours with 200 mL of each solvent. Acetone and 96% ethanol were evaporated at the rotary evaporator TURBOVAP 500 (Caliper Life Sciences Inc., Hopkinton, MA, USA). Then, these extracts were kept for 16 h in a chemical exhaust hood for optimal solvent evaporation. After filtration with filter paper, UBW was concentrated on a Rotavapor R-215 with a vacuum controller V-850 (BÜCHI Labortechnik AG, Flawil, Switzerland), and lyophilized with a freeze-dryer Christ Alpha 1-2L (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) connected to a vacuum pump RZ 2.5 (VACUUBRAND GmbH, Wertheim, Germany) [72]. All these dry extracts (dUBA, dUBE, dUBW) were transferred in sealed-glass containers and preserved in freezer (Sirge ® Elettrodomestici-S.A.C. Rappresentanze, Torino, Avigliana, Italy) at −18 • C [73] until processing.

HPLC-DAD Determination of Usnic Acid Content
A previously validated HPLC-DAD method was adapted for quantifying usnic acid [53].
The system has a Zorbax C18 analytical column 150 mm/4.6 mm; 5 µm (Agilent Technologies, Santa Clara, CA, USA). As a mobile phase, isocratic methanol: water: acetic acid = 80:15:5 was selected for 6 min per run, at an injection volume of 20 µL at a flow rate = 1.5 mL/min. The oven temperature was established at 25 • C, and the detection was performed at 282 nm.

Sample, Blank, Standard Solutions
All requested solutions were prepared using acetone as a solvent. The standard was usnic acid dissolved in acetone at concentrations of 2.5, 5, 10, 20, 50 µg/mL, with which the calibration curve ( Figure S3, Supplementary Materials) was drawn (y = 39.672x − 3.8228; R 2 = 0.999). Each dilution was injected 6 times (20 µL) in the chromatographic system, and the obtained retention time value was 4.463 ± 0.008 min.

Data Processing
Data processing was achieved using the Waters Empower 2 chromatography data software with ICS 1.05 (Waters Corporation, Milford, MA, USA).

HPLC-DAD Determination of Polyphenols
The polyphenols quantification was achieved using a standardized HPLC method. It was described by the USP 30-NF25 monograph and previously validated [74].

Equipment and Chromatographic Conditions
The Agilent HPLC-DAD system was the analytical platform, with the same Zorbax C18 column, 150 mm 4.6 mm; 5 µm. As a mobile phase, two solutions were used: solution A: 0.1% phosphoric acid and solution B: acetonitrile, with gradient elution, at 22 min per run, with the same injection volume and flow The temperature was set at 35 • C and the detection was performed at UV 310 nm.

Total Phenolic Content
The total phenolic content was determined using Folin-Ciocâlteu reagent through a spectrophotometric method detailed in a previous study [53]. Pyrogallol was selected as the standard, the TPC values being calculated as mg of pyrogallol equivalents (PyE) per gram extract.

DPPH Free-Radical Scavenging Activity Assay
The U. barbata extracts free radical scavenging activity (AA) was determined spectrophotometrically through the DPPH free-radical scavenging assay previously described [19].

Antibacterial Activity
The antibacterial effects were evaluated by an adapted disc diffusion method (DDM) from the Clinical and Laboratory Standard Institute (CLSI) [75], previously described [76].

Microorganisms and Media
We obtained all bacteria strains from the American Type Culture Collection (ATCC). Their identification was performed at the Department of Microbiology and Immunology, Faculty of Dental Medicine, Ovidius University of Constanta. The Gram-positive bacteria were S. aureus (ATCC 25923) and S. pneumoniae (ATCC 49619); the Gram-negative ones were Pseudomonas aeruginosa (ATCC 27853) and K. pneumoniae (ATCC 13883). As a culture medium for all bacterial strains, Mueller-Hinton agar was used.

Inoculum Preparation
We prepared the bacterial inoculum using the direct colony suspension method (CLSI). Thus, we obtained a 0.9% saline suspension of bacterial colonies selected from a 24 h agar plate, according to the 0.5 McFarland standard, with around 10 8 CFU/mL (CFU-colonyforming unit).

Lichen Samples Preparations
The fluid extracts were subjected to solvent evaporation in the rotary evaporator TURBOVAP 500. These concentrated extracts were kept for 2 h in a chemical exhaust hood for each optimal solvent evaporation. Then, all U. barbata extracts were redissolved in 0.1% DMSO [77], obtaining a final solution of 15 mg/mL concentration.
The dry lichen extracts were dissolved in 0.1% DMSO, resulting in 15 mg/mL concentration solutions.

Disc Diffusion Method
The 15 mg/mL lichen extracts in 0.1% DMSO were applied on Whatman ® filter paper discs (6 mm, Merk KGaA, Darmstadt, Germany). The negative control was the solvent (0.1% DMSO); UA of 15 mg/mL in 0.1% DMSO was the positive control for all extracts. We impregnated each filter paper disc with 10 µL control and sample solutions. The standard antibiotic discs (6 mm) with ofloxacin 5 µg and ceftriaxone 30 µg (Oxoid, Thermo Fisher Scientific GmbH, Dreieich, Germany) were selected for antimicrobial activity evaluation. These blank discs were stored in a freezer at −14 • C and incubated for 2 h before analysis at room temperature.
Each inoculum was applied over the entire surface of the plate with the suitable culture media using a sterile cotton swab. After 15 min of drying, the filter paper discs were applied to the inoculated plates; they were incubated at 37 • C for 24 h. 4.7.5. Reading Plates Circular zones of a microorganism growing inhibition around several discs could be observed, examining the plates after 24 h incubation. The results of the disc diffusion assay are expressed in the inhibition zone diameter (IZD) measured in mm. These IZD values quantify bacterial strains' susceptibility levels after 24 h incubation [78].

Interpretation of Disc Diffusion Method results
Usnic acid and U. barbata extracts' IZD were compared to the IZD values of the positive controls represented by the blank antibiotic discs, ofloxacin 5 ug and ceftriaxone 30 ug [78]. In DDM, IZD values inversely correlate with minimum inhibitory concentrations (MIC) from standard dilution tests. According to CLSI [78], the interpretive categories are as follows: susceptible ("S"), intermediate-dose-dependent susceptibility ("I"), and resistant ("R") [46].

Activity Index
The activity index (AI) [47] is calculated using the following formula: where IZD sample-inhibition zone diameter for each U. barbata extract, and IZD standardinhibition zone diameter for each antibacterial drug, used as standard.

Data Analysis, Software
All analyses were accomplished in triplicate, and the results are expressed as the mean (n = 3) ± SD, calculated by Microsoft 365 Office Excel (Redmond, Washington, DC, USA). The p-values were calculated with the one-way ANOVA test; when the p-value was <0.05, the differences between the obtained mean values were considered significant. The principal component analysis (PCA) [51] was performed using XLSTAT 2022.2.1. by Addinsoft (New York, NY, USA) [79].

Conclusions
Our study analyzed the phenolic constituents and bioactivities of six U. barbata lichen extracts obtained through two low-cost conventional techniques widely used in pharmaceutical laboratories. Despite the same ratio between the dried lichen and the solvent (w/v), all lichen extracts displayed significant differences regarding the phenolic metabolites' diversity and amount due to extraction conditions, with a substantial impact on their bioactivities. All U. barbata extracts show antiradical activity; the antibacterial study proves that the U. barbata extracts in acetone and ethanol obtained through both methods considerably inhibit bacterial colony growth. Both Gram-positive bacteria and P. aeruginosa of Gram-negative ones reveal the highest sensibility.
Our results suggest that further research could extend the antibacterial studies, exploring their effects on other bacteria species. Future studies could optimize both extraction processes to obtain U. barbata extracts with valuable bioactivities for potential pharmaceutical applications.