Antimicrobial Activity of Paepalanthus planifolius and its Major Components against Selected Human Pathogens

The chemical investigation of ethyl acetate extract from Paepalanthus planifolius capitula resulted in the identification of 1H-naphtho[2,3-c]pyran-1-one,9-[(6-O-β-D-glucopyranosylβ-D-glucopyranosyl)oxy]-3,4-dihydro-10-hydroxy-7-methoxy-3-methyl, semi-vioxanthin 9-O-β-D-glucopyranoside, toralactone-9-O-β-D-glucopyranoside, paepalantine9-O-β-D-glucopyranoside, semi-vioxanthin, 1H-naphtho[2,3-c]pyran-1-one,3,4-dihydro9,10-dihydroxy-5,7-dimethoxy-3-methyl, vioxanthin and paepalantine dimer, and also the isolation and identification of a new naphthopyranone dimer named planifoliusin A. The chemical structures of two compounds were elucidated by performing spectroscopic 1D and 2D nuclear magnetic resonance (NMR) experiments and spectrometric HRMS (high-resolution mass spectrometry) analysis. Other six naphthopyranone dimers were proposed by MS fragmentation patterns. The minimum inhibitory concentration (MIC) values for vioxanthin (7.8 μg mL), planifoliusin A (15.6 μg mL) and the ethyl acetate extract (31.2 μg mL) showed antimicrobial activity against Staphylococcus aureus (ATCC 25923).


Introduction
Staphylococcus aureus is a leading cause of bacterial infections in hospitals and communities worldwide, and is one of the most important agents responsible for healthcare-associated infections, it has been estimated that around 20-30% of the human population are carriers of S. aureus.The increased occurrence of methicillin resistant S. aureus (MRSA) strains has created genuine clinical and therapeutical problems. 1onsidering that the antimicrobial resistances have been raised as a concern and the necessity to search for new antimicrobials agents as potential candidates for the treatment of several bacteria and fungi species, it is even more relevant to discover antimicrobial drugs. 2 Eriocaulaceae are ornamental plants popularly known as "evergreens" and comprise about 1200 species in 10 genera. 3Among these genus, Paepalanthus has approximately 357 species which 95% are endemic species restricted to Espinhaço Range in Brazil. 4,5Although Eriocaulaceae are used in the manufacture of ornamental products, literature reports studies with Eriocaulaceae species that have promising antimicrobial activity. 6,7Other studies have previously demonstrated the importance of extract and isolated substances in the Eriocaulaceae family, mainly because they present a great biological potential, such as the already identified antioxidant activities, 8,9 cytotoxic, 10,11 antiulcerogenic, 12 antibacterial and vulvovaginal candidiasis treatment. 13,14hus, in search of new biological compounds we described the antimicrobial activity of ethyl acetate (EtOAc) extract of capitula from Paepalanthus planifolius and its phytochemical characterization.Furthermore, the antimicrobial activities of isolated compounds were evaluated.

Antimicrobial activities and identification of naphthopyranones in the extract
The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) for the antimicrobial activity of the capitula EtOAc extract were evaluated against four human pathogenic microorganisms: Escherichia coli (ATCC 25922), Salmonella setubal (ATCC 19196), Staphylococcus aureus (ATCC 25923) and Candida albicans (ATCC 10231).Antimicrobial activity is considered good if the extract MIC values displayed lower than 100 µg mL -1 ; moderate from 100 to 500 µg mL -1 ; weak from 500 to 1000 µg mL -1 ; and over 1000 µg mL -1 the extract is considered inactive. 15Then the EtOAc extract showed a moderate activity against the Gram-negative bacteria tested, which displayed MIC values of 500 µg mL -1 .For the Gram-positive bacteria, it was considered a good activity (MIC value of 31.2 µg mL -1 ), while for the yeast fluconazole-resistant C. albicans, it was shown a low activity (Table 1).Once that the extract showed promised activity against S. aureus, we performed its chemical study.
Naphthopyranones exhibit a wide biological activity, including antimicrobial, anti-inflammatory, mutagenic, cytotoxic properties, among others. 21Thus, it is possible to relate that the good antimicrobial activity presented by the EtOAc extract of P. planifolius comes from this major naphthopyranones identified in the extract.
Compound 9 was elucidated by nuclear magnetic resonance (NMR) spectra, including 1 H, 13 C, HSQC (heteronuclear single quantum correlation), HMBC (heteronuclear multiple bond correlation) and 1 H-1 H COSY (correlation spectroscopy), and showed some similarities when compared to paepalantine dimer and vioxanthin isolated from the Paepalanthus species. 18,20The 1 H NMR spectrum displayed four signals of aromatic hydrogens at    Comparing and contrasting the above information with the data derived from the analysis of the 2D NMR spectra, it was possible to conclude that 9 is a naphthopyranone dimeric with a monomeric lactone, similar to paepalantine and the other monomer similar to semi-vioxanthin. 18,19he 1 H-1 H COSY correlations indicated the sequence H-4 ↔ H-3 ↔ H-11 confirming the presence of semivioxanthin derivatives. 19In the HMBC spectrum, diagnostic long range correlations were observed for the unit derived from the semi-vioxanthin between H-5 with the carbons C-4, C-4a and C-5a.The unit of the paepalantine derivative was evident by the correlation of the H-4' with C-4a', C-3' and C-5'.Additional evidence was given in the HMBC spectrum of which correlations to 2 J and 3 J of the OH-9 ) made it possible to establish that the linkage between two monomeric units is between C-8/C-8'.Thus, the compound 9 was proposed to be made up by two different monomeric portions, semi-vioxanthin and paepalantine, linked through a C-8/C-8' bond (Figure 3).The CD (circular dichroism) spectrum of planifoliusin A (9) showed negative Cotton effect at 293 nm that corresponds to n → π* transition (CO lactone) (Figure 4).Comparison of this negative Cotton effect with (R)-semivioxanthin revealed the absolute configuration at C-3 of planifoliusin A to be R.    Vioxanthin (7) was identified by comparison of its 1 H NMR, 13 C NMR and MS data with literature values. 18dditionally, the CD spectrum of vioxanthin ( 7) allowed to confirm the (P,R,R)-vioxanthin. 23

Antimicrobial activities of isolated compounds
Planifoliusin A (9) and vioxanthin (7) showed the respective MIC values of 1000 and 500 µg mL -1 for E. coli; 500 and 1000 µg mL -1 for S. setubal; both compounds exhibited significant activity against S. aureus with MIC values of 15.6 (9) and 7.8 µg mL -1 (7); and MIC values above 1000 µg mL -1 for C. albicans (Table 1).It has been commonly described that plant extracts are more active against Gram-positive bacteria than against Gram-negative bacteria. 24The existing difference between them can be explained because the cell walls of Gram-negative bacteria are less permeable to antimicrobial metabolites. 25In addition, the results for the MBC showed that the EtOAc extract and the compounds 9 and 7 displayed bactericidal activity from 62.5 to 1000 µg mL -1 , while for S. aureus these compounds showed MBC of 62.5 (9) and 250 µg mL -1 (7), indicating bacteriostatic behavior of these compounds for S. aureus.
The compounds 9 and 7 displayed a good antibacterial activity for the strains tested and, together with the results of the EtOAc extract, have shown pronounced efficacy against the Gram-positive bacteria S. aureus.

Conclusions
The LC-ESI-MS n analysis established a method for identifying naphthopyranones in the EtOAc extract of capitula from Paepalanthus planifolius in which 15 compounds were characterized.This is the first report on naphthopyranones dimers in Paepalanthus by LC-MS and this methodology could be used to identify these naphthopyranones in other Paepalanthus species.
It is also suggested that the antimicrobial activity against Staphylococcus aureus shown by the EtOAc extract is due to the presence of naphthopyranones derivatives, including the new planifoliusin A (9) compound and vioxanthin (7).Future studies may be made to isolate the new naphthopyranones dimers proposed (10-15) to obtain spectroscopic data and their antimicrobial activities.

LC-MS analysis
For the LC-MS analysis, 2.0 mg of the extract was dissolved in 2 mL of methanol:water (7:3, v/v).For both the analyses, the solution was filtered through a 0.22 µm polytetrafluoroethylene (PTFE) membrane and aliquots of 10 µL were directly injected into the LC-MS system.
The LC-ESI-TOF-HRMS experiment was performed using a UFLC (Shimadzu, Japan) containing two LC20AD solvent pumps, a SIL20A HT auto sampler, a CTO20A column oven and a CBM20A system controller and a diode array detector (SPD-M20AV), coupled with a microTOF (Bruker Daltonics, USA) mass spectrometer.The analyses were performed under the following conditions: capillary voltage, 3.5 kV; capillary temperature, 220 °C; end plate offset voltage, 490 V; nebulizer gas pressure, 5.5 bar; dry gas (N 2 ), 10 L min -1 .Mass spectra were measured from m/z 50-1300 in positive ion mode.
The LC-ESI-IT-MS/MS analyses were performed using a UFLC (Shimadzu), coupled with an AmaZon SL ion trap mass spectrometer (Bruker).The ion trap acquisition parameters were as follows: capillary voltage, 3.5 kV; end plate offset, 500 V; nebulizer gas pressure, 60 psi; dry gas (N 2 ), 10 L min -1 ; dry gas temperature, 320 °C.CID fragmentation was achieved in auto-MS/MS mode.
The analysis was performed using a gradient mode, eluted with a water:methanol gradient (70-92% methanol, 45 min) containing 0.1% formic acid, the flow rate was 1.0 mL min -1 , RP-18 column (Knauer, Eurospher II 250.0 × 4.6 mm, 5 µm) along with the protective guard column (Phenomenex, 4 × 3 mm) and the column oven was set at 30 °C.The software Bruker Compass DataAnalysis 4.2 was used to control the system, for data collection, and processing.The mass analyzer was calibrated using a solution of sodium trifluoroacetic acid as the internal standard.

Extraction and isolation
The dried powder of the P. planifolius capitula (50.1 g) was extracted with ethyl acetate (EtOAc, 1 L) for 48 h at room temperature by maceration and the same process was repeated for five times until exhaustion of the plant material.The solution was evaporated to dryness in vacuo to give the EtOAc extract (1.7 g, 3.4%), which was stored at 4 °C.
A portion of the extract (200 mg) was solubilized in EtOAc (5.0 mL) using ultrasound for 10 min and then centrifuged for 15 min.The precipitate (14 mg) was separated from the supernatant.The supernatant was filtered and fractionated by gel permeation chromatography using a Sephadex LH-20 column and eluted with EtOAc to give 5 fractions which were analyzed by silica gel thin layer chromatography (TLC, chloroform:EtOAc, 1:1, v/v, organic phase).Fractions 3-5 were combined and further purified by semipreparative HPLC-DAD which led to the isolation of planifoliusin A (9, 2.5 mg) and vioxanthin (7, 11.2 mg).

Antibacterial activity and minimum bactericidal concentration (MBC)
The evaluation of the antibacterial activity and the minimal inhibitory concentration (MIC) were determined by the broth microdilution method, as described in the M7-A6 reference guideline of the Clinical and Laboratory Standards Institute. 26The biological activity was evaluated against the bacteria E. coli (ATCC 25922), S. setubal (ATCC 19196) and S. aureus (ATCC 25923), all the microorganisms were obtained from the American Type Culture Collection (Rockville, MD, USA).They are incubated in the Muller-Hinton broth for 24 h at 37 °C.These inoculums were standardized at 1.0 × 10 8 CFU mL -1 (corresponding to 0.5 McFarland standards) by adjusting the optical density to 0.10-0.15at 620 nm.The assay was carried out in 96-well microplates containing 80 µL of Muller-Hinton broth and 20 µL of standardized inoculum.
The extract and the substances 9 and 7 were dissolved in DMSO:H 2 O (2:8, v/v) to initial concentrations of 1000 µg mL -1 .A two-fold serial dilution was carried out in order to obtain concentration ranging from 7.81 to 1000 µg mL -1 and 100 µL of each concentration were added to 96-well microplates.Ampicillin was used as positive control while DMSO:H 2 O (2:8, v/v) was used as the negative control.The plates were incubated at 37 °C for 24 h.The assay was displayed in triplicate.
The MIC of the samples was detected using spectrophotometric reading at 595 nm and with the addition of 30 µL of resazurin solution (100 µg mL -1 ), incubated at 37 °C for 2 h.The growth of bacteria changes the blue dye resazurin into a pink.The color pink indicates positive growth, whereas the color blue indicates growth inhibition.MIC was defined as the lowest sample concentration which prevented this change and exhibited inhibition of microorganism growth.For the determination of minimal bactericidal concentration (MBC), a portion from each well that showed antibacterial activity was plated on Muller-Hinton agar and incubated at 37 °C for 24 h.The lowest concentration that showed no bacteria growth in the subcultures was used as the MBC. 6tifungal activity and minimum fungicidal concentration (MFC) The evaluation of the antifungal activity and the minimal inhibitory concentration (MIC) were determined by the broth microdilution method, as described in the M27-A3 reference guideline of the Clinical and Laboratory Standards Institute, with modifications. 27,28The biological activity was evaluated against the fluconazole-resistant Candida albicans (ATCC 10231).
The yeast strain was incubated in 100 µL of RPMI 1640 (adjusted to pH 7.0 with 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, 0.165 mol L -1 ) for 48 h at 37 °C.The inoculum of yeast was standardized at 5.0 × 10 6 CFU mL -1 (corresponding to 0.5 McFarland standards) by adjusting the optical density to 0.12-0.15at 530 nm.The assay was carried out in 96-well microplates containing 100 µL of RPMI 1640 followed by a two-fold serial dilution to obtain concentration ranges of 7.81-1000 µg mL -1 to the extract and the isolated compounds.Fluconazole and amphotericin B were used as a positive control while DMSO:H 2 O (2:8, v/v) was used as the negative control.The plates were incubated at 37 °C for 48 h.The assay was displayed in triplicate.
The MIC of the samples was detected using spectrophotometric reading at 595 nm after the addition of 20 µL triphenyl-tetrazolium chloride (TTC) solution (0.02 g mL -1 ) incubated at 37 °C for 2 h.Yeast growth changes the colorless TTC to red.MIC was defined as the lowest sample concentration that prevented this change and exhibited inhibition of microorganism growth.For the determination of minimal fungicidal concentration (MFC), a portion from each well that showed antibacterial activity was plated on Sabouroud agar and incubated at 37 °C for 48 h.The lowest concentration that demonstrated no yeast growth in the subcultures was used as the MFC. 6
(d H 9.74) with C-9a (d C 108.5) and OH-10 (d H 13.81) with C-10a (d C 99.5) were observed, positioning these hydroxyls in the semi-vioxanthin monomer.The hydroxyls belonging to the paepalantine unit were also observed by the correlations in the HMBC spectrum of the hydrogen signal of the hydroxyl OH-9' (d H 9.75) with C-8' (d C 108.6) and the OH-10' (d H 13.44) with C-10a' (d C 96.9).Therefore, what remains is to connect the monomer of the paepalantine and of the semi-vioxanthin.The non-observance of H-6/H-8 meta coupling and H-6'/H-8' coupling combined with the chemical shift of C-8 (d C 108.1) and C-8' (d C 108.6

Table 2 .
Naphthopyranones identified by LC-HRMS and LC-MS/MS of EtOAc extract from P. planifolius capitula a auto-MS/MS mode acquisition did not acquire the fragmentations.t R : retention time.