Metabolomic Proﬁling, Antioxidant and Antimicrobial Activity of Bidens pilosa

: Bidens pilosa L. (fam. Asteraceae) is an annual herb used globally in phytotherapy and each plant material or the whole plant have been declared to be effective. Therefore, the aim of the present study was to conduct metabolomic proﬁling of different plant materials, including the quali-quantitative composition of phenolic compounds. The intrinsic scavenging/reducing properties and antimicrobial effects of the extracts were assayed against numerous bacterial, Candida and dermatophytes species, whereas docking runs were conducted for tentatively unravelling the mechanism of action underlying antimicrobial effects. Oligosaccharide, disaccharide and fatty acids were present at higher concentrations in root rather than in the other plant parts. Monoglycerides were more abundant in stem than in the other plant parts, whereas peptide and diterpenoid were prominent in leaf and root, respectively. By contrast, amino acids showed very different distribution patterns in the four plant parts. Regarding the phenolic composition, appreciable levels of caftaric acid were found in most of the analyzed methanol extracts, that were also particularly efﬁcacious as antiradical and anti-mycotic agents against C. albicans and dermatophytes. The docking experiments also showed a micromolar afﬁnity of caftaric acid towards the lanosterol 14 α -demethylase, deeply involved in fungal metabolism. In conclusion, the present study corroborates the B. pilosa as a phytotherapy remedy against infectious disease.


Introduction
Plants are an important source of pharmacologically active secondary compounds, that can be used in medicine to maintain and improve human health and also to treat specific conditions or illnesses [1,2]. Among these compounds, phenolics are very common and easy to find in plants, and they have demonstrated beneficial advantages in terms of antioxidant and antimicrobial activities. In particular, antioxidant metabolites can be used against different oxidative stress-induced diseases [3], while the antimicrobial properties of such compounds can rehabilitate the clinical application of older antibiotics by improving their efficacy and, therefore, by preventing the development of resistance [4]. Bidens pilosa L. (fam. Asteraceae) is an annual herb native to South America that is spread worldwide, especially in tropical and subtropical regions [5]. B. pilosa L. is used globally in phytotherapy more, a mass spectrometry ultra-performance liquid chromatography mass spectrometry (UHPLC)-QTOF method, coupled with different multivariate data analyses such as principle component analysis (PCA), was applied to B. pilosa metabolome aiming at investigating the metabolomic variation among the different plant materials of the same species and at evaluating this species as a potential antioxidant and antimicrobial. A liquid chromatography coupled to diode array and mass spectrometer (HPLC-DAD-MS) analysis was also conducted for measuring the levels of phenolic compounds in the extracts, whereas the intrinsic scavenging/reducing properties were determined via colorimetric methods. Finally, a docking approach was carried out for unravelling the putative mechanisms underlying the observed antimicrobial effects.

Plants Material
The mature seeds of B. pilosa L. were collected in July 2018, at an altitude of 1500-1800 m in Yabaramba zone, Kicukiro district, Rwanda. The seeds were cleaned and sterilized with ethylic alcohol solution 70% for 1 min and washed thoroughly 3 times with sterile distilled water. The sterilised seeds were planted in a garden pot containing the sterilised garden soil with NPK 12:11:18:2. The plants full grown were separated into roots, leaves and stems. The plant materials were separated into 4 samples that are leaves, roots, stems and whole plants. Afterwards, they were dried in an autoclave at 40 • C. The dried plant materials separated in leaves, roots, stems and whole plants were finely grounded and macerated in methanol for 7 days at 20 • C (1:10 w/v). The resulting extracts were then filtered through Whatman GF/C filters (Sigma, Germany), and the solvent evaporated under reduced pressure (40 • C, 218 mbar) using a rotary vacuum evaporator (Rotavapor R-100, Büchi, Switzerland). The residue was kept at −20 • C until further use.

Untargeted LC-MS/MS-Based Metabolomics
Untargeted metabolomics was carried out by using ultra-performance liquid chromatography mass spectrometry (UHPLC)-QTOF employing a 1260 ultra-high-performance liquid chromatograph and a G6530A QTOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). An Agilen JetStream ionization source in positive and negative polarity was also used. LC separation was performed using an Ascentis Express Peptide ES-C18 Supelco column (2.1 × 750 mm, 2.7 µm) with a gradient elution of mobile phase A (water + 0.1% Formic Acid) and mobile phase B (Acetonitrile with 0.1% Formic Acid). LC gradient consisted of holding solvent (A/B: 98/2) for 2 min, then linearly converting to solvent (A/B: 40/60) for 5 min, linearly converting to solvent (A/B: 20/80) for 1 min and holding for 2 min, then linearly converting to solvent (A/B: 98/2) for 0.5 min, and holding for 3 min for re-equilibration. The flow rate and column temperature were set to 0.45 mL/min and 45 • C, respectively. The mass spectrometer was operated in iterative Data Dependent Acquisition mode (50-1000 m/z), with a nominal resolution of 40,000 FWHM (full width at half maximum) and in the extended dynamic range mode using 5 precursors per cycle with collision energies of 30 eV. Peak picking and alignment were performed by MS-DIAL (ver. 4.38) with the following parameters: accurate mass tolerance (MS1) tolerance, 0.01 Da; MS2 tolerance, 0.025 Da; maximum charge number, two; smoothing method, linear weighted moving average; smoothing level, 3; minimum peak width, five scans; minimum peak height, 1000; mass slice width, 0.1 Da; sigma window value, 0.5; MS2Dec amplitude cut off, 0; exclude after precursor, true; keep isotope until, 0.5 Da; relative abundance cut off, 0; top candidate report, true; retention time tolerance for alignment, Compound annotation was made comparing the experimental MS/MS spectra to those available in the NIST2020 Tandem Mass Spectral Library. An m/z window of 0.005 Da and a relative intensity threshold of 0.5 were selected as input parameters. Only the compounds with an identification score cut off >80% were retained for further analysis. Principle component analysis (PCA) and Heatmap were performed with MetoboAnalyst 5.0 for either annotated metabolites or ontology grouped metabolites. For PCA and Heatmap, samples were normalized by median, followed by pareto scaling.

Determination of the Antioxidant Activity
The antiradical activity was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method. Each sample was mixed with 900 µL of 100 mM Tris-HCl buffer, pH 7.4, and then added to 1 mL of 0.5 mM DPPH in methanol (250 µM in the reaction mixture). The control sample was prepared using methanol. Trolox was employed as a reference antioxidant substance. Absorbances of the mixtures were measured at 517 nm. The activity was calculated as IC 50 Trolox equivalent. All tests and analyses were run in triplicate and averaged. For ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical scavenging) assay, the procedure followed the method of Arnao et al. with some modifications. The stock solutions included 7 mM ABTS solution and 2.45 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 14 h at room temperature in the dark. The solution was then diluted by mixing 1 mL ABTS solution with 60 mL methanol to obtain an absorbance of 0.706 ± 0.01 units at 734 nm. Fresh ABTS solution was prepared for each assay. Plant extracts (1 mL) were allowed to react with 1 mL of the ABTS solution and the absorbance was taken at 734 nm after 7 min using a spectrophotometer. The ABTS scavenging capacity of the extract was compared with that of Trolox and the activity was calculated as IC 50 Trolox equivalent. All determinations were performed in triplicate. The antioxidant capacity of methanolic solutions was estimated according to the procedure described by Benzie and Strain with some modifications. Briefly, 900 µL of FRAP (ferric reducing antioxidant power) reagent, prepared freshly and warmed at 37 • C, was mixed with 90 µL of distilled water and 30 µL of test sample. The final dilution of the test sample in the reaction mixture was 1/34. The FRAP reagent contained 2.5 mL of a 10 mmol/L TPTZ solution in 40 mmol/L HCl plus 2.5 mL of 20 mmol/L FeCl 3 ‚6H 2 O and 25 mL of 0.3 mol/L acetate buffer, pH 3.6. The absorbance was measured at 593 nm against the blank after 4 min. Methanolic solutions of known Fe(II) concentrations in the range of 100-2000 µmol/L (FeSO 4 ‚7H 2 O) were used for calibration. FRAP value was calculated and expressed as mM Fe 2+ equivalent (FE) per 100 g sample using the calibration curve of Fe 2+ . All determinations were performed in triplicate. In this assay, antioxidant capacity was determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation. A stock solution of β-carotene/linoleic acid mixture was prepared as follows: 0.5 mg β-carotene (0.9 mM) was dissolved in 1 mL of chloroform, then 25 µL linoleic acid and 200 mg Tween 40 was added. Then, 100 mL distilled water saturated with oxygen (30 min, 100 mL/min) was added with vigorous shaking; 2.5 mL of this reaction mixture was dispensed into test tubes and 100 µL portions of the methanol extracts were added; the emulsion system was incubated for up to 24 h at room temperature under agitation. The same procedure was repeated with the synthetic antioxidant butylated hydroxytoluene (BHT) as positive control, and a blank. After this incubation period, absorbances of the mixtures were measured at 490 nm. The activity was calculated as % Antioxidant Activity (AA) using the following equation: %AA = 100 × [1 − (As0 − Ast)/(Ac0 − Act)]. As0 is the absorbance of sample at 0 min, Ast is the absorbance of sample at 4 h, Ac0 is the absorbance of control sample at 0 min, and Act is the absorbance of control sample at 4 h. All tests were run in triplicate and averaged.

HPLC-DAD-MS Determination of Phenolic Compounds
B. pilosa methanol extracts were analyzed for phenol quantitative determination using a reversed-phase high performance liquid chromatography coupled to diode array and mass spectrometer (HPLC-DAD-MS) in gradient elution mode. The separation was conducted within 30 min of the chromatographic run, starting from the following separation conditions: 0.23% formic acid, 93% water, 7% methanol, as previously described [25]. The separation was performed on InfinityLab Poroshell 120 reverse phase column (C 18 , 150 × 4.6 mm i.d., 2.7 µm) (Agilent Santa Clara, CA, USA). Column temperature was set at 30 • C. Quantitative determination of phenolic compounds was performed via DAD detector. The extract was also qualitatively analyzed with an MS detector in negative ion mode, with the sole exception of rutin that was analyzed in positive ion mode. MS signal identification was realized through comparison with standard solutions and MS spectra present in the MassBank Europe database. Quantification was done through 7point calibration curves, with linearity coefficients (R 2 ) > 0.999, in the concentration range 2-140 µg/mL. The limits of detection were lower than 1 µg/mL for all assayed analytes. The area under the curve from HPLC chromatograms was used to quantify the analyte concentrations in the extract.

Antimicrobial Tests
In vitro antimicrobial activity of n-hexan, ethyl acetate and methanol extracts from  [26]. MICs have been determined using concentrations of the dry extracts in the range 1-0.031 mg mL −1 , derived from serial two-fold dilutions in Mueller-Hinton Broth (MHB). For the preparation of bacterial suspensions (inocula), three to five colonies of the bacterial strains used for the test were picked from 24 h cultures on tryptic soy agar plates (TSA) and pre-grown overnight in Mueller-Hinton broth (MHB) to reach a cell density of approximately 1-2 × 10 8 CFU mL −1 (analogous to the 0.5 McFarland standard). Hence, bacterial suspensions were diluted in fresh MHB and added to the MIC dilution series to reach 5 × 10 5 CFU mL −1 in each tube. This was confirmed by plating serial dilutions of the inoculum suspensions on Mueller-Hinton Agar (MHA). The set-up included bacterial growth controls in wells containing 10 µL of the test inoculum and negative controls without bacterial inoculum. MIC end-points were determined after 18-20 h incubation in ambient air at 35 • C [27]. MIC end-points were defined as the lowest concentration of either B. pilosa extracts or ciprofloxacin that totally inhibited bacterial growth [27]. Each test was done in triplicate. Geometric means and MIC ranges were calculated. Susceptibility testing against yeasts and filamentous fungi was performed according to the CLSI M38 (CLSI 2018) and M38-Ed3 (CLSI 2017) protocols [27][28][29]. RPMI (Roswell Park Memorial Institute) 1640 medium (Sigma) with L-glutamine and without sodium bicarbonate, supplemented with 2% glucose (w/v), buffered with 0.165 mol L −1 morpholinepropanesulphonic acid (MOPS), pH 7.0, was used throughout the study.
The inoculum suspensions were prepared from 7-day-old cultures grown on Sabouraud Dextrose Agar (SDA; Difco) at 25 • C and adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.11 (Mac Farland standard). Filamentous fungi (microconidia) and yeast inoculum suspensions were diluted to a ratio of 1:50 in RPMI 1640 to obtain twice an inoculum size ranging from 0.2 to 0.4 × 10 4-5 CFU mL −1 . This was further confirmed by plating serial dilutions of the inoculum suspensions on SDA. MIC end-points (µg mL −1 ) were determined after 24 h (for yeasts) and 72 h (for dermatophytes) of incubation in ambient air at 30 • C (CLSI 2017, CLSI 2018). For the plant extracts, the MIC end-points were defined as the lowest concentration that showed total growth inhibition [30]. The MIC end-points for fluconazole were defined as the lowest concentration that inhibited 50% of the growth when compared with the growth control [28]. Geometric means and MIC ranges were determined from the three biological replicates to allow comparisons between the activities of plant extracts.

Bioinformatics
Docking calculations were conducted through the Autodock Vina of PyRx 0.8 software, as recently described [31]. Crystal structures of target protein were derived from the Protein Data Bank (PDB) with PDB ID as follows: 5TZ1 (lanosterol 14α-demethylase). Discovery studio 2020 visualizer was employed to investigate the protein-ligand nonbonding interactions.

Untargeted LC-MS/MS-Based Metabolomics
Using normalized peak intensity and principal component analysis (PCA) clustering of all annotated compounds, clear differences were observed in the metabolite profiles of the four plant parts (Figures 1 and 2; Tables 1 and 2). Although PCA analysis indicated that the different plant materials display similar metabolite profiles, oligosaccharides, disaccharides and fatty acids were found to be much more abundant in root than in the other plant parts. Monoglyceride fraction was particularly present in stem rather than in the other plant parts. By contrast, peptides and diterpenoids were found at higher levels in leaf and root, respectively, whereas amino acids showed very different distribution patterns in the four plant parts. In future experiments, to improve understanding of metabolic pathways and considering the complexity of compounds measured and their various physical and chemical properties, an internal standard for each ontology group could be used to address the differences between the extraction and ionization processes.     In the heat map columns (data reported in triplicate), red color indicates higher relative levels of metabolites, whereas the blue color suggests a minor content of them. Compound annotation was made comparing the experimental MS/MS spectra to those available in the NIST2020 Tandem Mass Spectral Library. An m/z window of 0.005 Da and a relative intensity threshold of 0.5 were selected as input parameters. Only the compounds with an identification score cut-off > 80% were retained for further analysis. Heatmap was performed with MetoboAnalyst 5.0 for either annotated metabolites or ontology grouped metabolites. Samples were normalized by median, followed by pareto scaling. In the heat map columns (data reported in triplicate), red color indicates higher relative levels of metabolites, whereas the blue color suggests a minor content of them. Compound annotation was made comparing the experimental MS/MS spectra to those available in the NIST2020 Tandem Mass Spectral Library. An m/z window of 0.005 Da and a relative intensity threshold of 0.5 were selected as input parameters. Only the compounds with an identification score cut-off > 80% were retained for further analysis. Heatmap was performed with MetoboAnalyst 5.0 for either annotated metabolites or ontology grouped metabolites. Samples were normalized by median, followed by pareto scaling.

Antimicrobial Effects
The antimicrobial effects of n-hexane, ethyl acetate and methanol extracts were compared with reference drugs and presented in Tables 3-5. Overall, clinical Gram-negative bacterial strains (PeruMyc 2, 3, 5 and 7) showed a somewhat lower susceptibility to plant extracts than that of Gram-positive ones. This was particularly true for the B. cereus strain PeruMycA 4, that showed the lowest MIC values ( Table 3). Regardless of the bacterial strain used, n-hexane extracts showed the lowest antibacterial activity (Table 3). Table 4 shows the MIC ranges and geometric means of plant extract and fluconazole against the yeast species tested. C. parapsilosis (YEPGA 6551) were the most sensitive yeast strain to plant extracts, with MIC ranges of <0.031-0.198 mg mL −1 , while C. albicans (YEPGA 6379) showed the least sensitivity to the plant extract.

Phenolic Profile
The HPLC analyses showed that gallic acid, caftaric acid, catechin, chlorogenic acid, epicatechin and caffeic acid were present in most of the analyzed plant materials ( Figure 3A-D), with the only exception of leaf methanol extract. Among the identified compounds, caftaric acid (peak #3 in the Figure 3A-C; retention time: 9.15 min.) was revealed to be the prominent phenolic compound, especially in the extract prepared from the whole plant (3.03 µg/mL).

Phenolic Profile
The HPLC analyses showed that gallic acid, caftaric acid, catechin, chlorogenic acid, epicatechin and caffeic acid were present in most of the analyzed plant materials ( Figure  3A-D), with the only exception of leaf methanol extract. Among the identified compounds, caftaric acid (peak #3 in the Figure 3A-C; retention time: 9.15 min.) was revealed to be the prominent phenolic compound, especially in the extract prepared from the whole plant (3.03 µg/mL).  Table 1, caftaric acid (peak #3), catechin (peak #4), chlorogenic acid (peak #5) and epicatechin (peak #6) were identified in the whole plant (A), stem (B) and root (C). In the leaf (D), the principal phenolics identified in the extract were: gallic acid (peak #1), chlorogenic acid (peak #2) and epicatechin (peak #3).

In Silico Experiments
The results of the in silico experiment highlight the capability of caftaric acid to interact with the active site of the enzyme with a micromolar affinity, through the formation of both hydrogen bonds and alkyl interactions (Figure 4). The affinity of the caftaric acid was compared to that of the reference drug ketoconazole that, as expected, shows a much higher (sub-micromolar) affinity compared to that of caftaric acid.   Table 1, caftaric acid (peak #3), catechin (peak #4), chlorogenic acid (peak #5) and epicatechin (peak #6) were identified in the whole plant (A), stem (B) and root (C). In the leaf (D), the principal phenolics identified in the extract were: gallic acid (peak #1), chlorogenic acid (peak #2) and epicatechin (peak #3).

In Silico Experiments
The results of the in silico experiment highlight the capability of caftaric acid to interact with the active site of the enzyme with a micromolar affinity, through the formation of both hydrogen bonds and alkyl interactions (Figure 4). The affinity of the caftaric acid was compared to that of the reference drug ketoconazole that, as expected, shows a much higher (sub-micromolar) affinity compared to that of caftaric acid.

In Silico Experiments
The results of the in silico experiment highlight the capability of caftaric acid to interact with the active site of the enzyme with a micromolar affinity, through the formation of both hydrogen bonds and alkyl interactions (Figure 4). The affinity of the caftaric acid was compared to that of the reference drug ketoconazole that, as expected, shows a much higher (sub-micromolar) affinity compared to that of caftaric acid.

Intrinsic Scavenging/Reducing Properties
Finally, the radical scavenging/reducing properties of the meth losa plant materials were assayed ( Table 6). The values of the DPPH comparison with the activity of the Trolox and the best activity is with a rather good mean value of 9.9 IC50 referred to the Trolox. activity of the stems with a mean value of 101.4, whilst the roots mean value (15.2). The values of the ABTS assay are reported in com tivity of the Trolox and analogously to the DPPH test the best acti leaves, with a rather good mean value of 15.4 IC50 referred to the Tro the activity of the stems with a mean value of 89.1, whilst the roots mean value (25.3). The FRAP assay shows the mM Fe(II)+ equival sample; interesting is the activity of the leaves, with a mean value lower are the values of the other extracts: plants > roots > stems with 10.1, respectively. In Beta Carotene/Linoleic acid assay values are ex oxidant activity. The extracts do not show pro-oxidant activity bu value of the leaves (44.7) and of the plant (37.4). The antioxidant acti than that of the leaves in all the tests carried out and the methanol order leaves > plants > roots > stems.

Intrinsic Scavenging/Reducing Properties
Finally, the radical scavenging/reducing properties of the methanol extracts of B. pilosa plant materials were assayed ( Table 6). The values of the DPPH assay are reported in comparison with the activity of the Trolox and the best activity is shown by the leaves with a rather good mean value of 9.9 IC 50 referred to the Trolox. Decidedly low is the activity of the stems with a mean value of 101.4, whilst the roots have a medium-low mean value (15.2). The values of the ABTS assay are reported in comparison with the activity of the Trolox and analogously to the DPPH test the best activity is shown by the leaves, with a rather good mean value of 15.4 IC 50 referred to the Trolox. Decidedly low is the activity of the stems with a mean value of 89.1, whilst the roots have a medium-low mean value (25.3). The FRAP assay shows the mM Fe(II)+ equivalent (FE) for an 100 g sample; interesting is the activity of the leaves, with a mean value of 73.

Discussion
Considering the traditional ethnopharmacology and phytotherapy uses [6,7], in the present study, different materials from B. pilosa have been assayed in order to unravel plant material composition and extracts' antimicrobial effects. Oligosaccharide, disaccharide and fatty acids were found to be much more abundant in root than in the other plant parts. Monoglycerides were more abundant in stem than in the other plant parts, whereas peptide and diterpenoid were more abundant in leaf and root, respectively. By contrast, amino acids showed very different distribution patterns in the four plant parts. The microbiological study investigated the potential anti-bacterial and anti-fungal effects of the extracts against selected pathogen strains. All tested extracts showed fungal growth inhibition; particularly active were the ethyl acetate and methanolic extracts from root and leaves that showed the highest antifungal activity among all samples tested. Ethyl acetate and methanolic extract were also classified as potent for dermatophyte when compared to n-hexane. The results clearly demonstrated that the extracts were less effective when compared to the reference drugs, namely the anti-bacterial ciprofloxacin and the anti-mycotic fluconazole and griseofulvin. Nevertheless, the ethyl acetate and methanol extracts of B. pilosa displayed anti-mycotic activity on C. albicans (YEPGA 6379) and dermatophytes; this deserves further investigation. Considering the results of the antimicrobial tests pointing to promising activity of polar extracts from B. pilosa as anti-bacterial and anti-mycotic agents, a quali-quantitative HPLC-DAD-MS analysis was conducted on phenolic acids and flavonoids from B. pilosa methanol extracts, in order to unravel the putative mechanisms underlying the observed antimicrobial effects. In this regard, it is sensitive to highlight that phenolic compounds could explain, albeit partially, the anti-mycotic effects induced by polar extracts [32,33]. Specifically, the HPLC analyses showed that gallic acid, caftaric acid, catechin, chlorogenic acid, epicatechin and caffeic acid were present in most of the analyzed plant materials. Caftaric acid is known to be a phytocompound characterizing Echinacea species [34]. However, different studies suggest the presence of this phenolic compound in Bidens species, including B. tripartita and B. pilosa [28,29,35,36], thus further corroborating the results of the present phytochemical investigation. A docking approach was also conducted in order to predict putative interactions between the caftaric acid and the lanosterol 14α-demethylase, playing a master role in fungal metabolism. The results of the in silico experiment highlight the capability of caftaric acid to interact with the active site of the enzyme with a micromolar affinity, through the formation of both hydrogen bonds and alkyl interactions. The putative affinity of caftaric acid towards the selected target enzyme was lower compared to that of the reference drug ketoconazole. However, this putative affinity is consistent with the concentration of the phenolic compound in the extract, and also with the extract MIC values, above all against the Candida species. Therefore, the present docking experiments highlight the importance of phenolic compounds in mediating, albeit partially, the antimicrobial effects induced by B. pilosa methanolic extracts. Finally, the radical scavenging/reducing properties of the methanol extracts of B. pilosa plant materials were assayed. The intrinsic antioxidant effects of the extracts were evaluated through ABTS, DPPH and Beta-Carotene assays. The antioxidant action of the stems is less than that of the leaves in all of the tests carried out and the methanolic extract follows the order leaves > plants > roots > stems. Antioxidants attract a growing interest owing to their protective roles against oxidative deterioration in food and in the body, and against oxidative stress-mediated pathological processes. Screening of antioxidant properties of plants requires appropriate methods, which address the mechanism of antioxidant activity and focus on the kinetics of the reactions including the antioxidants. Many studies evaluating the antioxidant activity of various samples of research interest using different methods in food and human health have been conducted. Methods based on inhibited autoxidation are the most suited for termination-enhancing antioxidants and for chain-breaking antioxidants. In general, the methods for the determination of the antioxidant capacity of plant extract can deactivate radicals by two major mechanisms and were divided into two major groups: assays based on the single electron transfer (SET) reaction, and assays based on hydrogen atom transfer (HAT). The end result is the same, regardless of mechanism, but kinetics and potential for side reactions are different. SET-based methods detect the ability of a potential antioxidant to transfer one electron to reduce any compound, including metals, carbonyls, and radicals [37]. HAT-based methods measure the ability of an antioxidant to quench free radicals by hydrogen donation [38]. For this purpose, the most common methods used in vitro determination of antioxidant capacity of plant raw extract were considered in this manuscript. The methanol extracts of roots, stems, leaves, and whole plants were tested with DPPH assay (1,1-diphenyl-2-picrylhydrazyl radical scavenging), ABTS assay (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical scavenging), FRAP assay (ferric reducing antioxidant power), and beta carotene/linoleic acid assay (double bond antioxidant power). Methods based on the HAT reaction include the β-Carotene bleaching assays [37]. The SET-based methods include the following assays: 2,2-Diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH·), ferric ion reducing antioxidant power assay (FRAP), and 2,2-Azinobis 3-ethylbenzthiazoline-6-sulfonic acid radical scavenging assay (ABTS). It was reported that ABTS methods used both HAT and SET mechanisms [38]. Phenolic compounds are secondary plant metabolites naturally present in almost all plant materials, including food products of plant origin. Many of the health-protective effects of phenolic compounds have been ascribed to their antioxidant, anticarcinogenic, antimutagenic, antimicrobial, anti-inflammatory and other biological properties [39,40]. The correlation matrix for Pearson coefficients provides a high correlation with the FRAP assay (0.90) and a very low inverse correlation with the linoleic assay (−0.53); intermediate values were for DPPH (−0.70) and ABTS (−0.74) assays. Flavonoids are cyclized diphenylpropanes that commonly occur in plants and particularly plant foods. They are polyphenolic compounds, which are very effective antioxidants that serve against chronic diseases. The intrinsic antioxidant properties have also been related to enzyme inhibition properties [41]. Flavonoids have been isolated from almost all parts of the plant such as leaves, stems, roots, fruits, or seeds. In general, the effective antioxidant ability of flavonoids depends on some factors: the metal-chelating potential that is strongly dependent on the arrangement of hydroxyls and carbonyl group around the molecule, the presence of hydrogen or electron-donating substituents able to reduce free radicals, and the ability of the flavonoid to delocalize the unpaired electron leading to formation of a stable phenoxy radical [39]. Similarly, the correlation matrix for Pearson coefficients provides a high correlation with the FRAP assay (0.88), a very low inverse correlation with the linoleic assay (−0.32), and intermediate values for DPPH (−0.64) and ABTS (−0.68) assays. The chemical complexity of the extracts, stemming from the fact that they are often mixtures of many compounds, with differences in functional groups, polarity, and chemical behavior, could lead to unpredictable results about their possible antioxidant activity.

Conclusions
The results of antimicrobial susceptibility testing were analyzed and thoroughly discussed, also with respect to results from similar studies. It should be born in mind, however, that comparisons between bioactivity results are always difficult, because working protocols may differ in terms of extraction methods, test organisms and test systems used [42]. In the present study, the microbiological assays pointed to the promising activity of polar extracts, namely methanol extracts, as anti-mycotic agents. The anti-mycotic effect could be partially mediated by phenolic compounds detected by colorimetric and HPLC-DAD-MS analyses. The pattern of phenolic compound composition could also explain the intrinsic scavenging/reducing properties of the methanol extracts. The present phytochemical determinations also validated previous studies suggesting the presence of caftaric acid, in the phytocomplex of B. pilosa [35]. Additionally, considering the intrinsic anti-inflammatory properties of caftaric acid [43], we cannot exclude its involvement in mediating, albeit partially, the anti-inflammatory effects of B. pilosa [44]. Therefore, future studies could be conducted in order to investigate anti-inflammatory effects induced by the present extracts from B. pilosa.