Gallomyrtucommulones G and H, New Phloroglucinol Glycosides, from Bioactive Fractions of Myrtus communis against Staphylococcus Species

Myrtaceae family is a continuous source of antimicrobial agents. In the search for novel antimicrobial agents against Staphylococcus species, bioactive fractions of Myrtus communis L., growing in the Sardinia island (Italy) have been investigated. Their phytochemical analysis led us to isolate and characterize four alkylphloroglucinol glycosides (1–4), three of them gallomyrtucommulones G–H (1,2), and myrtucommulonoside (4) isolated and characterized for the first time. The structures of the new and known compounds, endopreroxide G3 (5), myricetin-3-O-glycosides (6,7) were determined based on the spectroscopic evidence including 1D-/2D-NMR and HR-MS spectrometry. Enriched fractions as well as pure compounds were tested for their antimicrobial activity by broth micro-dilution assay against Staphylococcus epidermidis and S. aureus. Results reported herein demonstrated that gallomyrtucommulone G (1) showed a selective antimicrobial activity against both S. aureus strains (ATCC 29213 and 43300) until 16 μg/mL while gallomyrtucommulone D (3) showed the best growth inhibition value at 64 μg/mL.


Introduction
The skin is populated by different genera of bacteria and yeasts, coexisting in the same microenvironment thanks to interspecies interaction ensuring the health of the host [1]. Among the Gram-positive bacteria, the main constituent of the normal flora on the human skin and mucous membranes is Staphylococcus epidermidis, a coagulase-negative Staphylococcus that contributes to host defense by avoiding the colonization from other potentially harmful microorganisms. It also plays an important role in the modulation of inflammation by increasing the skin's antimicrobial defense [2]. Nevertheless, once the host epithelial barrier is compromised, S. epidermidis is responsible for nosocomial infection due to its ability to form biofilm on indwelling medical devices, causing prosthetic joint, vascular graft, surgical site, central nervous system shunt and cardiac device infections [3,4]. Moreover, the skin microbiota can also be colonized by pathogenic strains, such as S. aureus, the main cause of both community-and hospital-acquired infections. It can colonize almost every tissue and organ, as well as implants, and can frequently manifest resistance to antibiotic therapy, a serious problem that is involving researchers worldwide. The emergence of multi-drug resistant strains such as MRSA (methicillin-resistant S. aureus) is nullifying the efforts made until now, virtually eliminating the use of β-lactams as therapeutic options against S. aureus [5].
Among natural sources, plants represent a valuable source of a wide spectrum of specialized metabolites, which have a vast variety of biological activities including antimicrobial properties [6,7]. In this context, the Myrtaceae family, the ninth largest family of flowering plants and known for their high biodiversity, is a source of antimicrobial agents against pathogenic microorganisms [8,9]. Callistrilone A from Callistemon rigidus [10], rhodomyrtone A from Rhodormytus tomentosa [11], eugenials C and D from Eugenia umbelliflora [12], and myrtocummulone A from Myrtus communis L. [13] are the most common phloroglucinols derivatives that have been reported for their good activity against different strains of S. aureus MRSA. In particular, M. communis L. (Myrtaceae), a Mediterranean evergreen shrub growing in the wild in hill regions from Morocco to Iran, is an important source of myrtocummulones derivatives [14,15], while alkylphloroglucinol glycosides are less prevalent [16]. This species has been used since ancient times as an ornamental plant and as a source of fragrance and medications. Furthermore, M. communis has displayed anti-inflammatory, antiseptic, antimicrobial, and hypoglycemic properties [15]. In pursuing our investigation of plants growing in the Sardinia region [17], endowed of peculiar chemical profiles [18], the bio-guided phytochemical investigation of glycosidic fractions of M. communis leaves was performed. Herein we describe the isolation, from active fractions against Staphylococcus epidermidis, and the structural characterization by 2D-NMR of two new galloylated alkylphloroglucinol glucosides (Gallomyrtucommulones G-H, 1-2), one alkylphloroglucinol glucosides (Myrtucommulonoside, 4) along four known compounds. The antimicrobial properties of the enriched fractions and pure compounds have been also assessed.

Antimicrobial Activity of Hydroalcholic Crude Extract and First Purification Step
The crude extract of M. communis leaves (MC) was tested for antimicrobial capacities on two strains of S. epidermidis: S. epidermidis ATCC 12228 (control strain) and S. epidermidis ATCC 35984 (biofilm producer strain). MC showed a discrete antimicrobial activity on both strains resulting more active on S. epidermidis ATCC 35984. In fact, at 32 µg/mL MC reduced by 45.6% ± 4.8 the growth of the strain biofilm producer (ATCC 35984), while at the same concentration the inhibition of the growth of the control strain (ATCC 12228) was 26.7% ± 2.1. However, a discrete antimicrobial activity (31.3% ± 2.8) against S. epidermidis ATCC 35984 was evident until 2 µg/mL. The positive control, vancomycin, strongly inhibited the growth of S. epidermidis at 2 µg/mL (data not shown). By considering the promising results, MC was further chromatographed on Amberlite XAD-4 and the methanolic fraction was separated by CC-flash chromatography furnishing three fractions (MC_A-MC_C) from less polar MC_A to more polar MC_C. The fractions were characterized by different metabolites contents as highlighted by their 1 H-NMR profiling. In particular, MC_A showed prevailing signals in the aliphatic region (1-6-1.2 ppm) as six methyl singlets besides one singlet in the aromatic region. The 1 H NMR spectrum of MC_B fraction showed resonances typical of gallic acid (δ H 7.10) as well as a cluster of olefinic and oxymethine singlets in the range between 5.0 and 3.9 ppm, and a 6-acylated glycosyl moiety. The fine splitting of all signals in the upfield region of the spectrum suggested a mixture of other related compounds. Finally, MC_C was constituted almost exclusively by myricitrin [19].
All the three above mentioned fractions (MC_A-MC_C) were tested against both S. epidermidis strains ( Figure 1). Myricitrin turned out to be inactive (data not shown), while MC_A showed the best activity on S. epidermidis ATCC 35984, reducing the growth by 77%, until the concentration of 32 µg/mL. At the same concentration, MC_B reduced by only 25% the growth of the same bacterial strain. The similar trend has been revealed against S. epidermidis ATCC 12228. Based on the promising results, MC_A and MC_B were then chosen for a more in-depth phytochemical study.

Fractionation of MC_A: Antimicrobial Assessment
The interesting results obtained for the less polar fraction (MC_A) of crude extract prompted us to go on its purification. Three fractions, MC_A1, MC_A2 and MC_A3, were obtained and tested against both S. epidermidis strains until here used ( Figure 2). Only fraction MC_A3 turned out to be active. In fact, MC_A3 reduced by 70% the growth of S. epidermidis ATCC 12228 at 128 µg/mL, and against S. epidermidis ATCC 35984 induced a stronger percentage of growth inhibition (84.5%) at 16 µg/mL ( Figure 2). MC_A1 and MC_A2 did not inhibit the growth of S. epidermidis ATCC 35984.
Based on the interesting results obtained, all the fractions MC_A1, MC_A2, MC_A3, were assayed against the pathogenic strains of S. aureus ATCC 29213 and S. aureus ATCC 43300 (methicillin-resistant strain). The most active fraction turned out to be MC_A3, which showed the best activity against both S. aureus strains with a MIC value of 4 µg/mL ( Figure 2). The positive control, oxacillin, was not able to inhibit the growth of S. aureus MRSA as efficiently as MC_A3 did (data not shown). On the contrary, MC_A1 did not inhibit the growth of both strains, while MC_A2 turned out to be scarcely active, reducing by 45% the growth of S. aureus ATCC 43300 at 16 µg/mL ( Figure 2).
The activity of MC_A3 was also investigated in terms of bactericidal activity. The results, reported in Figure 3 (panel A), demonstrated the bactericidal activity at 8 and 4 µg/mL against both S. aureus strains, since it caused an over 3log 10 -fold reduction in the bacterial count (2.9 × 10 3 CFU/mL for 4 µg/mL treated-S. aureus ATCC 12228 versus 1.8 ×   To further investigate the antimicrobial activity of MC_A3, the synergistic interaction with oxacillin against MRSA was determined using the checkerboard technique. After 24 h of incubation, we analyzed the results to look for the best combination of MC_A3 and oxacillin. The total absence of growth was observed in the wells of the checkerboard plates with the combination between 2 µg/mL MC_A3 and 0.25 µg/mL oxacillin (MIC value for oxacillin alone was 10 µg/mL), with a decrease by half of the MIC value of MC_A3. The Fractional Inhibitory Concentration (FIC) value of 0.5 was indicative of an additive effect for MC_A3 and oxacillin. Moreover, 2D-NMR investigation of the most active last fraction MC_A3 revealed the presence of endoperoxide G3 as the main component together with ursolic acid [16] besides other aliphatic compounds present in trace. Further studies are, however necessary to assess if this activity can be ascribed to the single components or their mixture.

Fractionation of MC_B and NMR Characterization of Alkylphloroglucinol Glucosides
The MC_B fraction was purified by HPLC obtaining four related pure compounds (1-4, Figure 4), three of them isolated for the first time.  Compound 1 showed a molecular formula C 28 H 37 O 13 , which is in good agreement with the HRESI-MS and 13 C-NMR data (Table 1, Figure S1, see Supplementary Materials). The 1 H NMR spectrum of compound 1 showed typical resonances of a galloyl moiety as a singlet at δ H 7.07 (δ C 110.7).
An olefinic proton at δ H 5.08 (δ C 116.8), an oxymethine proton at δ H 3.90 (δ C 88.3), one allylic methyl (δ H 1.75), as well as four quaternary methyls at δ H 1.30, 1.22, 1.21, 1.19 were also evident in the 1 H NMR spectrum, besides other signals in the region between 3.00 and 4.60 ppm that suggested the presence of a sugar moiety. Finally, in the aliphatic region of 1 H NMR spectrum, the presence of a quartet at δ H 1.98 and of a triplet at δ H 0.93 suggested the presence of an ethyl moiety. The 13 C NMR showed 28 carbons identified, on the basis of the HSQC experiment ( Figure S2), as six methyls, two methylenes, nine methines, and eleven tetrasubstituted carbons, two of them identified as ketone carbonyls (δ C 210.8 and 216.4). The sequence of the glycosidic moiety was established based on heteronuclear two-bond correlations shown in the H2BC spectrum and the data, supported by the HSQC-TOCSY experiment, suggested the presence of a glucose moiety. The coupling constant value of the anomeric proton (J = 8.1 Hz) allowed a β configuration for the C-1 carbon to be assigned. The downfield shifted values of the H-6 doublet of doublets of glucose (δ H 4.51/4.47, δ C 65.2) and the correlations of these protons with the carboxyl carbon at δ C 168.8, allowed the localization of the galloyl group at the C-6 carbon of the sugar. The structure of metabolite 1 was confirmed by the HSQC and CIGAR-HMBC data, as reported in Table 1.  In the CIGAR-HMBC ( Figure 5B), the anomeric proton at δ H 4.58 showed cross peaks with the C-10 (δ C 88.3), in turn correlated with H-13 and H-14 methyls. These last methyls were heterocorrelated with the C-10 and the carbonyl carbon at δ C 216.4 (C-8) in turn heterocorrelated with the methyls 11 and 12. All these data (Table 1) are in agreement with the dihydroxylated cyclohexadione moiety [20] linked to a C5 unit. On the other hand, the presence of bands at 2930, 1742, 1731, 1605, cm −1 in the IR spectrum is in good accordance with data reported for related compounds isolated from M. communis [14]. The 1 H-spin system and 13 C multiplicities allowed us to characterize this latter unit: the cis configuration of the glycol moiety was assigned based on the observation of NOE among the vinylic proton H-4 and the oxymethine H-10 proton, the H-14 methyl and the methylene group ( Figure 5A). Finally, NOE between vinylic proton H-4 and the anomeric proton was also evident. This structure was confirmed by the ESI Q-TOF MS/MS analysis: the tandem MS analysis of the quasimolecular ion [M-H] − at m/z 581.2219 produced the fragment ion at m/z 169.0325, arising from the loss of galloyl moiety and one at m/z 124.0346, corresponding to the loss of CO 2 molecules from the latter. Another fragment ion at m/z 271.0609 is due to the cross-ring fragmentation of the galloyl-glucose moiety as already reported by Taamalli et al. (2013) [21] for gallomyrtucommulone C. Based on the analysis of spectroscopic evidence, the chemical structure of alkylphloroglucinol 1 has been elucidated and identified as gallomyrtucommulone G, structurally related to galloylated alkylphloroglucinol glucosides reported by Appendino et al. (2006) [20].  Figure S3). In fact, in compound 2, the methyl triplet and methylene protons resonated at δ H 0.97 and δ H 2.25, respectively (Table 1, Figure S4).
These last signals showed heterocorrelations with both olefinic carbon at δ C 118.5 (C-4) and δ C 150.4 (C-2) in turn heterocorrelated with allyl methyl at δ H 1.68. All others HMBC correlations ( Figure S5, Table 1) allowed to identify the compound 2 as gallomyrtucommulone G. It is worth mentioning that this is the third report that describes the presence of galloylated alkylphloroglucinol glucosides in plants belonging to Myrtaceae family. Besides the gallomyrtucommulones A-D reported from M. communis [20], related metabolites, namely gallomyrtucommulones E-F, were described as constituents of methanolic extract of Callistemon citrinus leaves and stems [16].
NMR data of compound 3 are in good agreement with those described for gallomyrtucommulone D, reported by Appendino et al. (2006) [20]. However, the careful inspection of NMR data, especially NOESY experiment (Figure 6), suggested a cis configuration of glycol moiety as indicated by observation of nOe correlations between the vinylic proton H-4 with oxymethine proton H-10, the allylic methyl (δ H 1.69) and the methyl singlet 14-CH 3 .   The NMR data of compound 5 are in good agreement with those reported in the literature for endopreroxide G3 [22], an antimalarial hormone presumably derived from the enolized trienone intermediate and so related with the gallomyrtucommulones G and H (1-2) described above. Finally, myricetin-3-O-rhamnoside (6) and myricetin-3-Ogalactoside (7), already reported as components of M. communis leaves [21], have been also isolated from MC_B fraction.

Fractionation of MC_B Antimicrobial Assessment
The compounds 1, 3 and 5 obtained from MC_B purification have been further investigated for their biological activities against both S. epidermidis and S. aureus (Figure 7). Compounds 2 and 4 were not tested due to insufficient material. Results highlighted that only gallomyrtucommulones G (1) and D (3) preserved their biological activity. In particular, the most active compound (1), provided strong activity against both S. aureus ATCC strains until 16 µg/mL (>65%, Figure 7). On the contrary, compound 3 showed the best activity only at 64 µg/mL (Figure 7) against MRSA (40%). Finally, both compounds turned out to be scarcely active on both strains of S. epidermidis, while endopreroxide G3 (5) was inactive.  The antimicrobial activity of MC_A3 and compound 1 turned out to be of great interest. We demonstrated that both were able to selectively reduce the growth of the pathogenic strains of S. aureus at a concentration that turned out to be ineffective on the skin microbiota component S. epidermidis. However, MC_A3 displayed a different action on the strain of S. epidermidis biofilm producer, since it was able to reduce bacterial growth even though at a higher concentration compared with S. aureus.
The results reported herein are of strong interest since the discovery of new molecules or enriched fractions selectively active on the skin microbiota components can pave the way for the realization of targeted therapy preserving the commensal bacteria, as S. epidermidis. Further studies could be addressed to investigate a possible involvement of these compounds in the regulation of the quorum sensing (QS) system accessory gene regulator (agr) within the Staphylococcal species [23,24].

Plant Material
Leaves of M. communis were collected at the flowering stage (April 2016) in the site of Cardedu (Sardinia, Italy, 39 • 49 52.3 N 9 • 20 31.1 E-693 m a.s.l.). A voucher specimen (CAG 514) has been deposited at the General Herbarium of the Department of Life and Environmental Sciences, University of Cagliari (Italy). M. communis is not protected by local or international regulations; therefore, no specific permission was required for its collection. The plant raw materials were dried in a ventilated stove at 40 • C to constant weight, powdered with liquid nitrogen, and stored at −20 • C until next analysis.

NMR Experiments
NMR spectra were recorded at 25 • C on 300.03 MHz for 1 H and 75.45 MHz for 13 C on a Bruker AVANCE II 300 MHz NMR Spectrometer Fourier transform in CD 3 OD or CDCl3 (Bruker, Billerica, MA, USA). Chemical shifts are reported in δ (ppm) and referenced to the residual solvent signal; J (coupling constant) is given in Hz. 1 H-NMR spectra were acquired over a spectral window from 14 to −2 ppm, with 1.0 s relaxation delay, 1.70 s acquisition time (AQ), and 90 • pulse width = 13.8 µs. The initial matrix was zero-filled to 64 K. 13 C-NMR spectra were recorded in 1 H broadband decoupling mode, over a spectral window from 235 to −15 ppm, 1.5 s relaxation delay, 90 • pulse width = 9.50 µs, and AQ = 0.9 s. The number of scans for both 1 H and 13 C-NMR experiments were chosen, depending on the concentration of the samples. With regard to the homonuclear and heteronuclear 2D-NMR experiments, the data points, number of scans, and increments were adjusted according to the sample concentrations. Correlation spectroscopy (COSY) spectra were recorded with gradient-enhanced sequence at spectral widths of 3000 Hz in both f2 and f1 domains; the relaxation delays were of 1.0 s. The total correlation spectroscopy (TOCSY) experiments were performed in the phase-sensitive mode with a mixing time of 90 ms. The spectral width was 3000 Hz. Nuclear Overhauser effect spectroscopy (NOESY) experiments were performed in the phase-sensitive mode. The mixing time was 500 ms, and the spectral width was 3000 Hz. For all the homonuclear experiments, the initial matrix of 512 × 512 data points was zero-filled to give a final matrix of 1 k × 1 k points. Protondetected heteronuclear correlations were also measured. Heteronuclear single-quantum coherence (HSQC) experiments (optimized for 1J (H,C) = 140 Hz) were performed in the phase-sensitive mode with field gradient. The spectral width was 12,000 Hz in f1 ( 13 C) and 3000 Hz in f2 ( 1 H), and had 1.0 s relaxation delay; the matrix of 1 k × 1 k data points was zero-filled to give a final matrix of 2 k × 2 k points. Heteronuclear 2 bond correlation (H2BC) spectra were obtained with T = 30.0 ms and a relaxation delay of 1.0 s; the third order low-pass filter was set for 130 < 1 J (C,H) < 165 Hz. A heteronuclear multiple bond coherence (HMBC) experiment (optimized for 1 J (H,C) = 8 Hz) was performed in the absolute value mode with field gradient; typically, 1 H-13 C gHMBC were acquired with spectral width of 18,000 Hz in f1 ( 13 C) and 3000 Hz in f2 ( 1 H) and 1.0 s of relaxation delay; the matrix of 1 k × 1 k data points was zero-filled to give a final matrix of 4 k × 4 k points. Constant time inverse-detected gradient accordion rescaled heteronuclear multiple bond correlation spectroscopy (CIGAR-HMBC) spectra (8 > n J (H,C) > 5) were acquired with the same spectral width used for HMBC. Heteronuclear single-quantum coherence-total correlation spectroscopy (HSQC-TOCSY) experiments were optimized for nJ (H, C) = 8 Hz, with a mixing time of 90 ms.

ESI MS Analyses
The exact masses were measured using a UPLC-QTOF ESI (Waters Xevo G2, Manchester, UK) high-resolution mass spectrometer (HRESI-TOFMS). Electrospray ionization in the negative polarity mode (ESI − ) was used with the following settings: sample probe capillary voltage 3000 V, sampling cone voltage 15 V; source temperature 120 • C, and desolvation temperature 250 • C. Desolvation and cone gas with flow rates of 850 and 10 L/h were used, respectively. The raw data files were processed using MassLynx

Susceptibility Assays on Bacteria Planktonic Cells
Minimal inhibitory concentrations (MIC) of the tested compounds were determined in specific medium by the broth micro-dilution assay, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST version 7.1, 7 June 2017), as previously reported [25]. Negative control wells were set to contain bacteria in Mueller-Hinton broth plus the amount of vehicle (ethanol) used to dilute each compound. Positive controls included vancomycin (2 µg/mL) and oxacillin (2 µg/mL). The MIC was defined as the lowest concentration of drug that caused a total inhibition of microbial growth after 24 h incubation time at 37 • C. Minimum bactericidal concentration (MBC) was obtained by plating 10 µL of the medium removed from the wells in the microtiter plates where no growth was observed after 24 h of incubation at 37 • C (MIC) and was then inoculated on tryptic soya agar (TSA, OXOID), incubated for further 24 h at 37 • C. MBC was defined as the minimum concentration of antimicrobial capable of inactivating more than 99.99% of the bacteria present, causing ≥ 3log10 reduction in colony count from the starting inoculum plated.

Live/Dead Imaging
To study the bactericidal activity of MC_A3, live/dead staining technique was used [26]. S. aureus MRSA were treated with MC_A3 at MIC concentration as above reported. Cells were harvested by centrifugation after 24 h and stained with solution of acridine orange and ethidium bromide (100 µg/mL) and kept in dark for 30 min. Cells were harvested and washed twice with PBS to remove the excess stains. The cells were then observed at 20× magnification (Iris Digita System, Twin Helix, Italy).

Checkerboard Method
The interaction between compound MC_A3 and oxacillin against MRSA was evaluated by the checkerboard method in 96-well microtiter plates containing Mueller-Hinton broth. Briefly, MC_A3 and oxacillin were serially diluted along the y and x axes, respectively. The final concentration ranged from 0.03 to 10 µg/mL for oxacillin and from 0.5 to 8 µg/mL (0.5, 1, 2, 4, 8 µg/mL) for MC_A3. The checkerboard plates were inoculated with bacteria at an approximate concentration of 10 5 × CFU/mL and incubated at 37 • C for 24 h, following which bacterial growth was assessed visually and the turbidity measured by microplate reader at 595 nm. The Fractional Inhibitory Concentration (FIC) index for each combination was calculated as follows: FIC index = FIC of MC_A3 + FIC of oxacillin, where FIC of MC_A3 (or oxacillin) was defined as the ratio of MIC of MC_A3 (or oxacillin) in combination and MIC of MC_A3 (or oxacillin) alone. The FIC index values were interpreted as follows: ≤0.5, synergistic; >0.5 to ≤1.0, additive; >1.0 to ≤2.0, indifferent; and >2.0, antagonistic effects [27].

Conclusions
Here we report the bio-guided investigation of glycosidic fractions of M. communis growing in the Sardinia island. This experimental approach led us to isolate and characterize for the first time four alkylphloroglucinol glycosides, three of them gallomyrtucommulones G-H (1-2), and myrtucommulonoside (4). Despite the wide distribution of oligomeric nonprenylated phologlucinols in Myrtaceae, the results reported herein represent only the third report regarding the presence of galloylated alkylphloroglucinol glucosides in plants belonging to Myrtaceae family. From a biological point of view, the results are of strong interest. We demonstrated that MC_A3 and 1 display a selective antimicrobial activity within the Staphylococcal genus, able to contrast the pathogenic species at very low concentrations but leaving undisturbed the commensal species of S. epidermidis. Due to their selectivity, we can hypothesize a possible use of these molecules as topical antibacterials for superficial skin infections.