Phytochemical Investigation of Myrcianthes cisplatensis: Structural Characterization of New p-Coumaroyl Alkylphloroglucinols and Antimicrobial Evaluation against Staphylococcus aureus

Species of Myrtaceae Juss., the ninth largest family of flowering plants, are a valuable source of bioactive specialized metabolites. A leading position belongs to phloroglucinol derivatives, thanks to their unusual structural features and biological and pharmacological properties. Myrcianthes cisplatensis (Cambess.) O. Berg, a common tree on the banks of rivers and streams of Uruguay, southern Brazil, and northern Argentina, with aromatic leaves, is known as a diuretic, febrifuge, tonic, and good remedy for lung and bronchial diseases. Despite knowledge about traditional use, few data on its phytochemical properties have been reported in the literature. The methanol extract of M. cisplatensis, grown in Arizona, USA, was first partitioned between dichloromethane and water and then with ethyl acetate. The enriched fractions were evaluated using a broth microdilution assay against Staphylococcus aureus ATCC 29213 and 43300 (methicillin-resistant S. aureus (MRSA)). The potential antimicrobial activity seemed to increase in the dichloromethane extract, with a MIC value of 16 µg/mL against both strains. Following a bio-guided approach, chromatographic techniques allowed for isolating three coumarin derivatives, namely endoperoxide G3, catechin, and quercitrin, and four new p-coumaroyl alkylphloroglucinol glucosides, named p-coumaroylmyrciacommulone A-D. Their structures were characterized through spectroscopic techniques: 2D-NMR experiments (HSQC, HMBC, and HSQC-TOCSY) and spectrometric analyses (HR-MS). The antimicrobial assessment of pure compounds against S. aureus ATCC 29213 and ATCC 43300 demonstrated the best activity for p-coumaroylmyrciacommulone C and D with the growth inhibition of 50% at 32 µg/mL against both strains of S. aureus.


Introduction
Myrcianthes cisplatensis (Cambess.) O. Berg is a native plant to northern Argentina, southern Brazil, and Uruguay, found at elevations of sea level to ca. 1000 m; it frequently grows along streams. It possibly grows in Paraguay, but no specimen records have been found. The genus Myrcianthes, a group of about 40 species, widely distributed in South America, is characterized by having an embryo with thick unfused plano-convex cotyledons and uniflorous or dichasial inflorescences [1]. Its fruits are edible berries eaten by birds and mammals and are used for the preparation of marmalades [2,3]. The genus belongs to the large family of Myrtaceae, which comprises ca. 5700 species with concentrations of diversity in South America and Australasia (WCSP) [4].

Antibacterial Assessment of Methanolic Extract and First Purification Step
The crude extract of M. cisplatensis (MYR) was tested for its antimicrobial potential against S. aureus ATCC 29213 and S. aureus ATCC 43300 (methicillin-resistant strain (MRSA)) using a broth microdilution assay. The extract showed promising activity on both strains, reporting the same inhibitory effects. MYR strongly reduced the growth of both strains (about 83%) at 64 µg/mL; a good antimicrobial activity was kept until the concentration of 16 µg/mL (Figure 1). The positive control, vancomycin, strongly inhibited the growth at 2 µg/mL (88.8% ± 2.1), whereas oxacillin was not able to inhibit the growth of the MRSA. The negative control methanol did not affect the cell growth at any of the tested dilutions.
Based on these promising results, MYR was further purified through liquid-liquid separation ( Figure 2) first with dichloromethane (MYR_A) and then using ethyl acetate (MYR_B). The medium-polar fraction (MYR_A) and the more-polar one (MYR_B) were characterized using different metabolite contents, confirmed by their 1 H-NMR profiling. In particular, MYR_A showed prevailing overlapped signals in the aliphatic region (1.6-0.2 ppm). Initially, the presence of fatty acids (Figure 2A) made it difficult to understand what kind of compounds were present in MYR_A. On the other hand, the 1 H-NMR spectrum of MYR_B was characterized by resonances typical of catechin ( Figure 2B). In addition, in the aromatic region (7.5-6.0 ppm), the presence of 1 the H-NMR spectrum of two additional meta-coupled doublets at δ H 6.36 and δ H 6.20, as well as the overlapped signals in the region between 5.0 and 3.3 ppm suggested the presence of flavonol glycosides in MYR_B. and ATCC 43300 strains. One-way ANOVA with Tukey multiple dicate significant differences among groups for each strains ( a p < Based on these promising results, MYR was further p separation ( Figure 2) first with dichloromethane (MYR_A (MYR_B). The medium-polar fraction (MYR_A) and the m characterized using different metabolite contents, confirm In particular, MYR_A showed prevailing overlapped sign 0.2 ppm). Initially, the presence of fatty acids (Figure 2A) m what kind of compounds were present in MYR_A. On the trum of MYR_B was characterized by resonances typical o tion, in the aromatic region (7.5-6.0 ppm), the presence of additional meta-coupled doublets at δH 6.36 and δH 6.20, as in the region between 5.0 and 3.3 ppm suggested the pres MYR_B.  and ATCC 43300 strains. One-way ANOVA with Tukey multiple comparisons: different l dicate significant differences among groups for each strains ( a p < 0.01 and b p < 0.05).
Based on these promising results, MYR was further purified through liquid separation ( Figure 2) first with dichloromethane (MYR_A) and then using ethyl (MYR_B). The medium-polar fraction (MYR_A) and the more-polar one (MYR_ characterized using different metabolite contents, confirmed by their 1 H-NMR p In particular, MYR_A showed prevailing overlapped signals in the aliphatic regi 0.2 ppm). Initially, the presence of fatty acids (Figure 2A) made it difficult to und what kind of compounds were present in MYR_A. On the other hand, the 1 H-NM trum of MYR_B was characterized by resonances typical of catechin ( Figure 2B). tion, in the aromatic region (7.5-6.0 ppm), the presence of 1 the H-NMR spectrum additional meta-coupled doublets at δH 6.36 and δH 6.20, as well as the overlapped in the region between 5.0 and 3.3 ppm suggested the presence of flavonol glyco MYR_B.  The two fractions were tested for their antimicrobial properties. MYR_A showed the best activity on both S. aureus strains, reducing the growth by 87% at the lowest concentration of 16 µg/mL ( Figure 2C), thus leading to more promising results with respect to the parental extract MYR. By contrast, MYR_B was strongly active at 128 µg/mL, reducing the growth of both Staphylococci strains by 84% (±2) but gradually decreased in activity at lower concentrations, being lowest at 16 µg/mL with the growth inhibition of 43% (± 1) and 30% (±1) against S. aureus 29213 and MRSA, respectively ( Figure 2C).

Purification of MYR_A and NMR Characterization of p-Coumaroyl Alkylphloroglucinols
A metabolic complexity of MYR_A required purification in order to perform the complete structural characterization of this medium-polar fraction. The HPLC, RP-18 led to the isolation of four p-coumaroyl alkylphloroglucinols (1-4, Figure 3), isolated for the first time in addition to endoperoxide G3 (5) and three coumarin derivatives (6)(7)(8). The two fractions were tested for their antimicrobial properties. MYR_A showed the best activity on both S. aureus strains, reducing the growth by 87% at the lowest concentration of 16 µg/mL ( Figure 2C), thus leading to more promising results with respect to the parental extract MYR. By contrast, MYR_B was strongly active at 128 µg/mL, reducing the growth of both Staphylococci strains by 84% (±2) but gradually decreased in activity at lower concentrations, being lowest at 16 µg/mL with the growth inhibition of 43% (± 1) and 30% (±1) against S. aureus 29213 and MRSA, respectively ( Figure 2C).

Purification of MYR_A and NMR Characterization of p-Coumaroyl Alkylphloroglucinols
A metabolic complexity of MYR_A required purification in order to perform the complete structural characterization of this medium-polar fraction. The HPLC, RP-18 led to the isolation of four p-coumaroyl alkylphloroglucinols (1-4, Figure 3), isolated for the first time in addition to endoperoxide G3 (5) and three coumarin derivatives (6)(7)(8). Compound 1, named p-coumaroylmyrciacommulone A, was described for the first time. It showed a molecular formula C29H38O11 in accordance with the pseudo-molecular [M + K] + and [M + Na] + peaks in the ESI Q-TOF HRMS at m/z 610.21 and m/z 585.23, respectively, and with the 13 C-NMR data. In fact, this spectrum showed 29 carbons identified, on the basis of an HSQC experiment (Table 1), as 6 methyls, 1 methylene, 13 methines, and 9 quaternary carbons, including 2 ketones (δC 211.2 and 216.7) and 1 ester (δC 169.1). The 1 H-NMR spectrum (Table 1)   Compound 1, named p-coumaroylmyrciacommulone A, was described for the first time. It showed a molecular formula C 29 H 38 O 11 in accordance with the pseudo-molecular [M + K] + and [M + Na] + peaks in the ESI Q-TOF HRMS at m/z 610.21 and m/z 585.23, respectively, and with the 13 C-NMR data. In fact, this spectrum showed 29 carbons identified, on the basis of an HSQC experiment (Table 1), as 6 methyls, 1 methylene, 13 methines, and 9 quaternary carbons, including 2 ketones (δ C 211.2 and 216.7) and 1 ester (δ C 169.1). The 1 H-NMR spectrum (Table 1) was dominated by the signals of the p-coumaroyl moiety suggested by the presence of an AA'BB' system, as two aromatic doublets at δ H 6.81 (H-2"/H-6", δ C 116.9) and δ H 7.46 (H-3"/H-5", δ C 131.0), as well as two olefinic doublets at δ H 7.63 (H-7"δ C 146.8) and δ H 6.35 (H-8"δ C 114.6). The ion at m/z 147.04, present in the ESI Q-TOF MS/MS spectrum, confirmed the presence of this moiety. The coupling constant value of 15.9 Hz for olefinic protons was in good agreement with an E configuration for the double bond of p-coumaroyl moiety. 145 118.7 (CH) 5.10 s 1,2,5 1,12,10 118.6 (CH) 5.10 s 1,2,5 1,10,12,14
The sequence of the glycosidic moiety was established based on the two-bond heterocorrelations evident in the H2BC spectra and also supported by the HSQC (Figure 4A) data, suggesting the presence of a glucose moiety. The β configuration of C-1 carbon was determined on the basis of the coupling constant value (J = 8.1 Hz) of the anomeric proton. The downfield-shifted values of methylene carbon (C-6 ), as well as of the H-6 doublet of the doublets of glucose (δ H 4.51/4.37 and δ C 64.2) and the correlations of these latter with carboxyl carbon at δ C 169.1, highlighted in the HMBC experiment ( Figure 4B), allowed the localization of p-coumaroyl moiety at the C-6 carbon of the sugar.   In the HMBC experiment ( Figure 4B), the anomeric proton at δ H 4.60 showed heterocorrelations with C-10 (δ C 89.1) methine carbon, whose proton at δ H 3.95 showed heterocorrelations with both ketone carbons C-6 (δ C 211.2) and C-8 (δ C 216.7), as well as with C-7 and C-9 quaternary carbons (δ C 48.7 and δ C 57.3, respectively) and with C-5 quaternary carbinol (δ C 83.4), confirming the presence of two hydroxyls on the cyclohexadione skeleton [16], while the hetero-correlation with C-4 methine at δ C 118.7 allowed us to localize the isobutylene moiety at the C-5 of cyclohexadione ( Figure 3).
The cis configuration of vicinal hydroxyl groups was determined based on the observed NOE, in the NOESY experiment, among the H-4 vinyl proton and both the H-10 proton and H-14 ( Figure S1). Moreover, the NOE between the H-10 proton and H-14 methyl allowed us to deduce a β orientation for C-10. All these spectroscopic data confirmed the hypothesized structure for p-coumaroylmyrciacommulone A (1). Compound 2 was identified as p-coumaroylmyrciacommulone B, namely a Z-isomer at the double bond of p-coumaroyl moiety of compound 1. It showed a molecular formula C 29 H 38 O 11 , in good agreement with the quasi-molecular ion [M − Na] + at m/z 585.23 and the fragment of p-coumaroyl moiety ion at m/z 147.04. The NMR spectra of compounds 2 and 1 showed the same 1 H spin system and 13 C multiplicities but showed significant differences for the olefinic protons of the p-coumaroyl moiety. In fact, the upfield-shifted values of protons H-8" at δ H 5.78 (δ C 116.4) and H-7" δ H 6.88 (δ C 145.7) were evident in the 1 H-NMR spectrum. Furthermore, the coupling constant value (J = 12.3 Hz) for olefinic protons H-8" and H-7" was in accordance with a cisgeometry for a double bond. These latter protons showed hetero-correlations with carboxyl carbon C-9" a δ C 168.2, which were, in turn, hetero-correlated with methylene protons H-6 (δ H 4.49/4.32) of the glucose unit. The hetero-correlation between the anomeric proton H-1 and carbon C-10 (δ H 3.91) suggested the presence of a dihydroxy cyclohexadione skeleton, whose C-10 was linked to a glucose-6" p-coumaroyl unit (Table 1, Figure 5B).  The cis configuration of vicinal dihydroxyl was determined based on the observation of the NOE between the oxymethine proton H-10 and vinylic proton H-4, as well as between the latter and methyl protons H-12 and H-14. Finally, the NOE was also evident between the oxymethine proton H-10 and the anomeric proton ( Figure 5A).

p-Coumaroylmyrciacommulone C (3) p-Coumaroylmyrciacommulone D (4)
Compound 4 showed similar 1 H spin systems and 13 C multiplicities ( Table 2) to those of compound 3. In particular, the coupling constant value for the olefinic proton at δ H 5.78 (J = 12.8) defined compound 4 as a cis isomer of compound 3. All these spectroscopic data (Table 2) confirmed the hypothesized structure for p-coumaroylmyrciacommulone D (4), described for the first time in the present paper.

The 2D-NMR Characterization of MYR_B
A 2D-NMR investigation was helpful in the characterization of the flavonoid compound of the more polar fraction MYR_B. In particular, NMR data allowed us to confirm the presence of catechin (9) [19]. Remaining to define were the second main flavonoidic compound. The 2D-NMR data suggested the presence of quercetin moiety as aglycon linked to rhamnose unit thanks to the long-range heterocorrelations ( Figure S3) between the anomeric proton at δH 5.36 (H-1") and a quaternary carbon compound at δC 134.0 (C-3). All these data allowed for the characterization of this compound as a 3-O-rhamnosyl quercetin (10).
Pure 9 and 10, obtained via MYR_B, were tested for their antimicrobial activity against S. aureus 25923 and MRSA.

Antimicrobial Assessment of Pure Compounds
Pure compounds 1-4, 6, and 9-10 were investigated for their antimicrobial properties against S. aureus ATCC 29213 and ATCC 43300 ( Figure 6). Catechin (9), quercitrin (10), and 5,7-dihydroxy-4-methylcoumarin (6) were inactive (data not shown). By contrast, p-coumaroyl alkylphloroglucinol (2-4) inhibited growth in the concentration range of 32 µg/mL to 128 µg/mL ( Figure 6). In particular, the best antimicrobial activity was shown by compounds 3 and 4, evaluated in the mixture, with the growth inhibition of 50% at 32 µg/mL against both S. aureus strains. The antimicrobial activity of compound 2 was also of great interest, reducing growth at 128 µg/mL (53% ± 4 and 55% ± 4) and 64 µg/mL (55% ± 4, and 51% ± 4) against ATCC 29213 and MRSA, respectively. By contrast, compound 1 reported weak activity only on S. aureus ATCC 29213 at 32 µg/mL. This result might reflect the chemical structures of the tested compounds. It was interesting to notice that the E/Z isomers 1 and 2 affected S. aureus growth differently. In fact, the best activity was reported by compound 2, which presented a cisconfiguration at the double bond of p-coumaroyl moiety. The literature data confirm these results. In fact, a double-bond geometry influences the antimicrobial properties of different etherolenic acid isomers against phytopathogenic bacteria with the Z isomer more active than the E isomer [20]. In addition, fatty acids with cis double bonds have more pronounced antibacterial properties than fatty acids with trans double bonds [21,22]. So, the potential of compound 2 might depend on this geometric isomerism. Furthermore, the antimicrobial results of compounds 2, 3, and 4 were in good agreement with the antibacterial activity reported for the related galloylated alkylphloroglucinol glucosides [14,16], whose moderate effect was reached in the concentration range of 64 µg/mL to 256 µg/mL. M. cisplatensis is not protected by local or international regulations; therefore, no specific permission was required for its collection. The leaves were dried in a ventilated stove at 40 • C to constant weight, powdered with liquid nitrogen, and stored at −20 • C until the next analysis.

General Chromatographic Procedures
Analytical TLC was performed on Merck Kieselgel 60 F254 or RP-8 F254 plates of 0.2 mm layer thickness. The plate was visualized using UV light or by spraying with H 2 SO 4 /AcOH/H 2 O (1:20:4), followed by heating at 120 • C for about 1 min. The plates were then heated for 5 min at 120 • C. Preparative TLC was performed on Merck Kieselgel 60 F254 plates, of 0.5 or 1 mm film thickness. Column chromatography (CC) was per-

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 CDCl 3 (Bruker, Billerica, MA, USA). Chemical shifts are reported in δ (ppm) and referenced to the residual solvent signal; J (coupling constant) is reported 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. The 13 CNMR spectra were recorded in the 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 was 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 a gradient-enhanced sequence at spectral widths of 3000 Hz in both f2 and f1 domains; the relaxation delays were 1.0 s. 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, an initial matrix of 512 × 512 data points was zero-filled to give a final matrix of 1 k × 1 k points. Proton-detected heteronuclear correlations were also measured. Heteronuclear singlequantum coherence (HSQC) experiments (optimized for 1 J (H,C) = 140 Hz) were performed in the phase-sensitive mode with field gradient. The spectral width was 12,000 Hz in f1 200 rpm. Each tested compound was dissolved in methanol (1 mg/200 µL) to give a stock solution and diluted in Mueller-Hinton (MH).

Susceptibility Assay on Bacteria Planktonic Cells
Minimal inhibitory concentrations (MICs) of the tested compounds were determined in a specific medium using the broth microdilution assay, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST version 7.1, 7 June 2017), as previously reported [14]. Briefly, 100 µL of bacterial culture was diluted in MH broth at a final cell concentration of 1 × 10 6 CFU/mL and added to each well of a 96-well plate containing 100 µL of MH with serially two-fold diluted compounds ranging from 128 to 16 µg/mL. Each MIC value indicates the lowest concentration of the compound that causes a total inhibition of bacterial growth after 24 h of incubation at 37 • C. Negative control wells were set to contain bacteria in the Mueller-Hinton broth plus the amount of vehicle (methanol) used to dilute each compound. The positive controls included vancomycin (2 µg/mL) and oxacillin (2 µg/mL).

Statistical Analysis
All the results of antimicrobial activity are expressed as the means ± standard deviation (S.D.). The results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc comparison tests to verify the differences between compounds and concentrations (p < 0.05).

Conclusions
The bio-guided phytochemical method is still the basic commonly accepted procedure for characterizing new natural products that are pharmacologically active. Following this approach, natural compounds with potent antimicrobial activity were isolated from the plants belonging to the Myrtaceae family. In particular, alkylphloroglucinols are responsible for the documented antimicrobial properties of the family. In this context, here, we reported the results of a phytochemical investigation of the methanol leaf extract of M. cisplatensis growing in Arizona. This led, for the first time, to the isolation and characterization of four p-coumaroylmyrciacommulones A-D. Unlike the wide distribution of alkylphloroglucinol in plants belonging to the Myrtaceae family, alkylphloroglucinol glucosides are reported as constituents of only few Myrtaceae plants. From both phytochemical and biological points of view, these results are of great interest since they pave the way for future studies. In particular, with docking molecular analysis, we plan to pursue an investigation of more active alkylphloroglucinols in order to provide some information about their interaction mechanism.

Conflicts of Interest:
The authors declare no conflict of interest.