Acylphloroglucinols from the fern Elaphoglossum lindbergii

Four new acylphloroglucinols have been isolated from a diethyl ether extract of the rhizomes and roots of the fern Elaphoglossum lindbergii . Their structures were elucidated by extensive analysis of spectroscopic data and comparison with those previously reported for other Elaphoglossum and Dryopteris acylphloroglucinols. These compounds showed mild antibacterial activity and altered biofilm formation of the Gram (+) bacterium Staphylococcus aureus at 100 μg/mL.


Introduction
In Argentina, the genus Elaphoglossum is represented by seven species: E. piloselloides (C.Presl) T. Moore, E. gayanum (Fée) T. Moore, E. yungense de la Sota, E. crassipes (Hieron.)Diels, E. lorentzii (Hieron.)H. Christ, E. lindbergii (Mett.3][4] In the last decades, attention has been drawn to these compounds due to their various biological activities, such as antidepressant, 5 antibacterial, 6 and molluscicidal. 2 As part of our ongoing investigations on Elaphoglossum species, we examined the diethyl ether (Et2O) extract of E. lindbergii to isolate 4 new acylphloroglucinols.The antibacterial and antibiofilm activity of these compounds was evaluated against Staphylococcus aureus.
Rhizomes and roots of E. lindbergii were air-dried, ground, and extracted with Et2O.The extract was fractionated by CC on silica gel and the fraction containing acylphlorolgucinols (TLC detection) was further purified by normal phase HPLC to afford four new prenylated acylphloroglucinols that were named lindbergins A-D (1-4).A high resolution measurement of the molecular ion of lindbergin A (1) gave m/z 556.3043, pointing to the molecular formula C32H44O8 (calculated 556.3037).The 1 H and 13 C NMR spectra of 1 (Tables 1 and 2) resembled those of elaphogayanin B, previously isolated from E. gayanum. 3The NOESY spectrum of 1 showed crosspeaks between the -OCH3 group and H-5′ and H-8′, revealing that the methoxy group was located at C-4′ instead of at C-6′ as in elaphogayanin B. Key H-C long range correlations of the hydroxyl protons, the methylene bridge protons, and H-5′ with the ring carbons were observed in the HMBC spectrum of lindbergin A (1), allowing the unambiguous assignment of the ring carbons (Table 3).As previously observed for other phloroglucinol derivatives, 2,3 the 5-OH hyroxyl proton shows HMBC correlations with C-14 and C-15, indicating that this proton is shared by the oxygen atoms at C-14 and C-5 forming a six-membered ring by hydrogen bonding.These crosspeaks are useful to identify the acyl group located at C-6 in acylphloroglucinol derivatives that possess an acylfilicinic acid-type ring.Full assignment of 1 H and 13 C spectra was accomplished by analysis of 1 H 1 H COSY, HSQC and HMBC spectra.Based on the forementioned evidence, the structure of 1 was established as shown.
Lindbergin B (2) showed a molecular ion peak [M] + at m/z 804.4091 in its HRFABMS spectrum that was consistent with the molecular formula C46H60O12 (calculated 804.4086).An AB system signal accounting for the 2 CH2 bridges was observed in the 1 H NMR spectrum of 2 centered at  3.56, indicating that both bridges are attached to an aromatic and an acylfilicinic acid-type ring. 7For lindbergin B, the 1 H NMR signals were broad, as previously observed for acylphloroglucinols containing more than two rings (Figure 1). 7The 13 C NMR spectrum showed duplicated or broad signals for the acylfilicinic acid-type ring carbons, suggesting the presence of two of such rings.Evidence for three acyl groups ( 209.6, 207.8, and 207.7) was obtained from the 13 C NMR spectrum of 2 (Table 2), confirming the presence of three rings, since in fern phloroglucinol derivatives, each ring only carries one acyl group.The acyl groups of 2 were identified as two butanoyls and one hexanoyl.3][4] This empirical observation can be useful to identify such residues in phloroglucinols containing three or more rings.The location of the acyl groups was established through HMBC correlations observed between 5-OH and both C-14 and C-14′′.From the above evidence, the structure of linbergin B (2) was deduced as depicted.
Table 1. 1 H NMR data of compounds 1-4 (acetone-d6, 500 MHz) Signals in a column marked with the same letter may be exchangeable.
The molecular formula of lindbergin C (3), C48H64O12, was deduced from its HRFABMS (observed m/z 832.4409, calculated 832.4399).Its 1D NMR data (Tables 1 and 2) resembled those of 2, but, in the 1 H NMR spectrum of compound 3 signals for one butanoyl and 2 hexanoyl residues were detected.The location of the acyl groups was established through long range H-C correlations.Thus, the structure of lindbergin C (3) was assessed as shown.
Lindbergin D (4) has a molecular formula C50H68O12 as deduced from a molecular ion peak at m/z 860.4736 in its HRFABMS spectrum (calculated 860.4712).The NMR traces of 4 resembled those of 2 and 3 but, in the case of 4, the presence of 3 hexanoyl residues was clearly observed.Accordingly, the structure of lindbergin D (4) was elucidated as illustrated.
The molecular formula of lindbergin C (3), C48H64O12, was deduced from its HRFABMS (observed m/z 832.4409, calculated 832.4399).Its 1D NMR data (Tables 1 and 2) resembled those of 2, but, in the 1 H NMR spectrum of compound 3 signals for one butanoyl and 2 hexanoyl residues were detected.The location of the acyl groups was established through long range H-C correlations.Thus, the structure of lindbergin C (3) was assessed as shown.Lindbergin D (4) has a molecular formula C50H68O12 as deduced from a molecular ion peak at m/z 860.4736 in its HRFABMS spectrum (calculated 860.4712).The NMR traces of 4 resembled those of 2 and 3 but, in the case of 4, the presence of 3 hexanoyl residues was clearly observed.Accordingly, the structure of lindbergin D (4) was elucidated as illustrated.

Effects on biofilm
Biofilms, which are aggregations of sessile bacteria surrounded by a polymeric matrix, 8 are the leading cause of chronic nosocomial infections.Diseases such as endocarditis, osteomyelitis and medical device-related infections are caused by S. aureus biofilms and are not readily treatable with antibiotics.In fact, biofilms are resistant to antibiotic levels 10 up to 1,000-fold higher than planktonic or free-floating bacteria. 8The aim of this research was to determine the effects of compounds 1-4 on S. aureus biofilm and to analyze their mode of action.
Lindbergin B (2) considerably inhibited biofilm production by S. aureus (82 % at 100 g/mL, Figure 2).At lower concentrations (50, 25, and 12.5 g/mL) no significant difference with the control was detected.As the reduction in bacterial growth was 86% at the mentioned dose, apparently, the observed inhibition in biofilm formation might simply be a consequence of reduced growth.It is important to point out that the antibiotic azithromycin (positive control) produced 76 % reduction of biofilm at 25 g/mL.Acylphloroglucinols affect bacterial growth and biofilm formation in different ways.While lindbergin B (2) reduced both, yungensins A, B, and D-F, previously isolated from E. yungense, 4 stimulated biofilm production but reduced bacterial growth.It is believed that lipophilic compounds intercalate in bacterial membranes, altering their fluidity. 9Bacteria then produce a hydrophilic exopolysaccharide that constitutes a physical barrier to the intercalation, as an adaptation strategy to a hostile environment. 9This phenomenon might be useful to explain the stimulation of biofilm formation produced by compound 4 (14 %), that carries several lipophilic moieties, as well at that of yungensins A, B, and D-F, 4 that carry one or two geranyl groups.The intercalation of these compounds inside the membrane would greatly alter membrane fluidity leading to cell lysis.
In addition, lindbergins A-D, produced no significant disruption of mature biofilm compared to control at 50, 25, and 12.5 g/mL.

Antibacterial activity
Acylphloroglucinols 1-4 show mild antibacterial acitivity against an ATCC collection strain of S. aureus with MICs > 100 g/mL in all cases.At 100 g/mL, 45, 86, 41 and 35 % growth inhibitions were detected after 24 h incubation, respectively.It is noteworthy that compound 2 strongly inhibited bacterial growth in the first 11 h of (OD < 0.05).

Experimental Section
General.Optical rotations were measured on a JASCO P-1030 polarimeter.IR spectra were recorded by the diffuse reflectance method on a Shimadzu FT/IR-8400S spectrophotometer.Low and high resolution mass spectra were registered in the positive mode on a JEOL JMS AX-500 spectrometer. 1 and 2D NMR experiments were performed with standard pulse sequences and parameters on a Varian Unity 500 using acetone-d6 as solvent and internal reference.Column chromatography (CC) was carried out over silica gel (70-230 mesh) with an n-hexane-EtOAc gradient as eluent.Preparative high pressure liquid chromatography (HPLC) was performed on a Gilson instrument equipped with a Chemcopak silica gel column (Chemcosorb 5 Si-U, 5 μm, 250 × 10 mm i.d.).CFU approximately.Mueller-Hinton (MH) medium was employed to prepare the inoculum and the dilutions of the samples.The control contained the solvent mixture (EtOH-EtOAc) used to dissolve the samples (the final concentrations of EtOH and EtOAc did not exceed 0.7 % and 0.3 %, respectively).Plates were incubated at 37 °C during 24 h.Growth was detected as turbidity (600 nm) relative to its record at the beginning of the experiment.Measurements were performed using a microtitre plate reader (Power Wave XS2, Biotek, Vermont, USA).The assay was carried out in eight replicates for each compound.Azithromycin (25 μg/mL) was used as positive control.Biofilm experiments. 11The effect of compounds 1-4 on biofilm production by S. aureus ATCC 6538 P was evaluated at 100, 50, 25, and 12.5 μg/mL.The test was performed by the crystal violet staining method.After 24 h of incubation of the strain in the presence of the evaluated compound, the amount of biofilm was measured by staining bound cells with crystal violet (1% w/v) for 20 min.Then, the medium was discarded, the wells were rinsed with H2O (x2), the dye was dissolved in 200 μL of EtOH, and absorbance was recorded at 540 nm using a microtitre plate reader.Eight replicates were performed for each sample.Disruption of mature biofilm was evaluated after two hours of incubation in presence of the tested compounds (50, 25 and 12.5 μg/mL).An overnight culture of S. aureus, 20 μL, was placed on each well.A dilution of the sample to be tested in the culture medium (MH) was added to reach a final volume of 200 μL.After 2 h, the biofilm was quantified as described in the previous paragraph.

Figure 1 .
Figure 1.Part of the 1 H NMR spectra of compounds 1 and 2 (500 MHz, acetone-d6).** Note that for lindbergin B (2), containing three rings, some 1 H NMR signals are broadened in comparison with those of lindbergin A (1) and their multiplicity is almost completely undetected.
Significant differences in mean values compared to control (Tuckey test).

Figure 2 .
Figure 2. Effect of 100 g/mL of compunds 1-4 on biofilm production by S. aureus ATCC 6538 P evaluated through absorbance measurements (540 nm) employing the crystal violet staining method.

Table 1 .
( a Overlapping signals, b Signals may be exchangeable, c Obscured.