Antimicrobial Activity of Piper gaudichaudianum Kuntze and Its Synergism with Different Antibiotics

One of the oldest forms of medical practice is the use of plants for the treatment and prevention of diseases that affect humans. We have studied the antimicrobial activity and synergism of Piper gaudichaudianum Kuntze with different antibiotics. The crude extract from the leaves of P. gaudichaudianum was submitted to chromatographic separation, resulting in five fractions. Fraction F3 contained a chromone (2,2-dimethyl-6-carboxycroman-4-one), and fraction F2 contained isomers that are prenylated derivatives of benzoic acid [4-hydroxy-(3',7'-dimethyl-1'-oxo-octa-E-2'-6'-dienyl)benzoic acid and 4-hydroxy-(3',7'-dimethyl-1'-oxo-octa-2'-Z-6'-dienyl) benzoic acid]. The chemical structures of both compounds were determined by analysis of 1H-NMR, 13C-NMR, COZY, DEPT, HMQC, and HMBC spectral data, and by comparison with data in the literature. The crude extract, fraction F2, and fraction F3 showed good activity against Staphylococcus aureus, Bacillus subtilis, and Candida tropicalis. The two benzoic acid derivatives only showed activity against S. aureus and B. subtilis. The bioauthographic analysis showed an inhibition zone only in fraction F2. Fractions F2 and F3 showed synergism in combination with ceftriaxone, tetracycline, and vancomycin. Morphological changes in form and structure were found by scanning electron microscopy in S. aureus treated with the combination of fraction F2 with vancomycin.

isolated from plants of the genus Berberis, inhibit the efflux pump in S. aureus, thereby boosting the antibacterial alkaloid berberine [13]. Piperine, the alkaloid found in plants of the family Piperaceae, has synergistic activity in combination with ciprofloxacin against S. aureus, including MRSA strains [14]. Totarol, also a phenolic diterpene, showed activity in inhibiting the efflux pump of S. aureus [15]. The purpose of this study was to isolate the chemicals present in the leaves of P. gaudichaudianum, and to evaluate their antibacterial activity and synergism with other antibiotics against S. aureus.
A previous report by Silva and co-workers [17] showed that the hydroalcoholic extract and the amides piperovatine and piperlonguminine from P. ovatum Vahl have good antimicrobial activity against B. subtilis and C. tropicalis. Another study reported the antibacterial activity of both crude extract and purified active compound of Piper regnellii, traditionally used in Brazilian folk medicine to treat infectious diseases [18] The compound that showed antibacterial activity against methicillinresistant S. aureus (MRSA) was identified as eupomatenoid-5 by spectroscopic analysis.
Fraction F4 (ethyl acetate) showed moderate activity against C. tropicalis with MIC 500 μg/mL, and was inactive against the others. Fraction F5 (methanol) was inactive against all microorganisms tested.
Purified substances FDA 47-58 and FDB 9 showed activity against the bacteria S. aureus and B. subtilis, with MIC of 31.25 μg/mL and 12.5 μg/mL for S. aureus, respectively, and 7.81 μg/mL and 6.25 μg/mL for B. subtilis respectively.
Although the bioautography test showed an inhibition zone only against S. aureus for the F2 fraction, the minimum inhibitory concentration test showed that the EB, fraction F3 and F1, also possessed activity against S. aureus (Tables 1 and 2). The low MICs could explain the absence of the inhibition of the other fractions and EB in the assays.
In general, secondary metabolites of plants are a source of bioactive substances, and scientific interest in these has been increasing during the search for new drugs, as well as new centers that can serve as model compounds. Chromones are not as abundant in Piper species as are amides, lignoids, phenylpropanoids and terpenes, but these substances are rich in pharmacological potential. Chromones in immature and mature fruits of P. gaudichaudianum show activity against E. faecium, M. luteus, and S. faecium [6]. Salazar et al. [16] found antifungal activity of chromones of leaves and stems of Peperomia villipetiola against Cladosporium cladosporioides and C. sphaerospermun. Chromones isolated from Peperomia serpens also showed antifungal activity [20]. Other chromones obtained through synthesis have shown activity against MRSA [21]. Chromones and chromanones isolated from a methanol extract of Hypericum sikokumontanum showed activity against Helicobacter pylori in a study by Tanaka et al. [22]. Piper hostmannianum and Piper aduncum yielded a benzoic-acid derivative 3-(2-hydroxy-3-methyl-3-butenyl)-4-methylhydroxybenzoate with molluscicidal activity [23,24]. From Piper lanceaefolium, prenylated derivatives of benzoic acid that had antifungal activity were isolated [25]. Piper multiplinervium accumulates a derivative of benzoic acid with antimicrobial activity against S. aureus, E. coli, Klebisiella pneumoniae, Mycobacterium smegmatis, P. aeruginosa, and C. albicans [26]. These studies indicate a tendency for chromones and derivatives of benzoic acid to show antimicrobial activity, as also found in this study ( Table 2).
The synergism of the crude extract, fraction F2, and ejection fraction F3 with antibiotics of different classes was verified through the checkerboard technique and the isobologram (Figure 4). The MIC of ceftriaxone was 6.25 μg/mL when the drug was tested alone, and decreased to 1.56 μg/mL in the presence of 100 μg/mL of fraction F3. For vancomycin, MIC was 1.25 μg/mL when tested alone, and decreased to 0.31 μg/mL in the presence of 50 μg/mL of fraction F2. For tetracycline, the MIC was 0.39 μg/mL when tested alone, and decreased to 0.09 μg/mL in the presence of 100 g/mL of fraction F3. Several studies of natural products have demonstrated the effect of the combination of drugs against microorganisms. Stermitz et al. [13] demonstrated the synergistic activity of porphyrin compounds and flavonolignans with the alkaloid berberine; the compounds inhibited the efflux pump of S. aureus, potentiating the antibacterial effect of berberine. Compounds such as diterpenes isolated from Rosmarinus officinalis and Lycopus europaeus showed a synergistic effect with erythromycin against resistant strains of S. aureus [27][28][29][30].

General
The NMR spectra were obtained in a Bruker ARX400 (9.4 T) and Varian Gemini 300 (7.05 T) instruments, using deuterated solvent for field homogeneity, TMS as internal standard and temperature constant of 298 K. IR: film NaCl plates; ES-MS were recorded on a Micro-mass Quattro LC, HRMS: Auto-spec Micro-mass EBE and EI-MS on a CG/EM-SHIMADZU QP 2,000 A. CC: silica gel 60 (70-230 and 230-400 mesh); TCL: silica gel plates F254 (0.25 mm in thickness).

Plant Extraction and Purification
The leaves (720 g) of the P. gaudichaudianum were extracted with ethanol-water (9:1 v/v, 7L) for 48 h at room temperature. The solvent was removed under vacuum at 40 °C to give an aqueous extract and a dark green residue. The aqueous extract from the crude hydroalcoholic extract was lyophilized (16.4 g) and the residue from crude extract in glass bottle was washed with dichloromethane. The organic solvent was removed to give the dichloromethane extract (11.87 g), which was chromatographed in a vacuum silica-gel apparatus, eluted with gradients of hexane, dichloromethane-

Thin Layer Chromatography
Kieselgel GF 254 plates, 20 × 20 cm, 1 mm thick, were used. Plant extracts (1 μg/mL) were applied (50 µL) and the chromatogram developed using hexane-ethyl acetate (50:50) as solvent. TLC plates were run in duplicate and one set was used as the reference chromatogram. Spots and bands were visualized by UV irradiation (245 and 366 nm) and Godin's Reactive (solution of 1% of vanillin and perchloric acid in a 1:1 proportion). The other set was used for bioautography. Amikacin (12.8 µg, Bristol Myers Squibb) was used as reference antibiotic.
Bacteria were maintained on Mueller Hinton Agar and sub cultured in Mueller Hinton Broth before each experiment. Yeasts were maintained at 4 °C on Sabouraud Dextrose Agar plates and sub cultured at 37 °C in Sabouraud Dextrose Broth before each experiment, to ensure viability and purity. The dermatophyte, was maintained on Sabouraud Dextrose Agar slants at 10 °C and sub cultured monthly throughout this study.

Bioautography
Chromatograms developed as described above were placed in a square plate with cover and inoculums of S. aureus containing 10 6 CFU/mL in molten Mueller-Hinton agar was distributed over the place. After solidification of the medium, the TLC plate was incubated overnight at 37 °C. Subsequently the bioautogram was sprayed with an aqueous solution of 2,3,5-triphenyltetrazolium chloride (TTC) and incubated at 37 °C for 4 h. Inhibition zones indicated the presence of active compounds.

Antibacterial Susceptibility Testing
The minimum inhibitory concentrations (MICs) of all compounds and reference antibiotics were determined by micro dilution techniques in Mueller Hinton broth for bacteria, and Sabouraud broth for yeasts, described by the Clinical and Laboratory Standards Institute (CLSI) [34]. Inoculates were prepared in the same medium at a density adjusted to a 0.5 McFarland turbidity standard (10 8 colonyforming units [CFU]/mL) and diluted 1:10 for the broth micro dilution procedure. Micro titer trays were incubated at 37 °C, and the MICs were recorded after 24 h for bacteria and 48 h for yeast of incubation. For dermatophyte the period of incubation time was 72 h at 28 °C. Two susceptibility endpoints were recorded for each isolate. The MIC was defined as the lowest concentration of compounds at which the microorganism tested did not demonstrate visible growth. Minimum bactericidal concentration (MBC) and Minimum fungicidal concentration (MFC) were defined as the lowest concentration yielding negative subcultures or only one colony.

Scanning Electron Microscopy
To investigate the effect of fraction F2 and vancomycin alone and in combination on the morphology of S. aureus, treated and control cells were examined by scanning electron microscopy. Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, and small drops of the fixed cells were placed on a specimen support with poly-L-lysine for 1 h at room temperature. Subsequently, the samples were dehydrated in a graded ethanol series, critical-point dried in CO 2 , coated with gold, and examined under a Shimadzu SS-550 scanning electron microscope ( Figure 5).

Checkerboard
From the results of the minimum inhibitory concentration tests, the combination of EB, F2, and F3 was done with the antibiotic ceftriaxone, chloramphenicol, penicillin, tetracycline, and vancomycin using the "checkerboard" method against the bacterium S. aureus. The combinations of EB and fractions with the antibiotics were tested in Mueller-Hinton Broth with an inoculum of 1 × 10 4 CFU/mL of S. aureus and different concentrations of the drug, ranging from 500 to 3.13 µg/mL, and antibiotics, ranging from 25 to 0.01 µg/mL. In a 96-well plate, each well received 100 μL of HCM, except column 12A, which received 200 μL of the antibiotic to be tested. The remaining wells of column 12 received 100 μL of each antibiotic, followed by serial dilutions to column 2. Then, the wells of the line received 100 μL of EB or fractions, followed by serial dilutions up to row G. Row H and column 1 were the controls for the antibiotic and EB/fractions, respectively. Each well contained a combination of different concentrations of EB and fractions with antibiotics. The plates were incubated at 37 °C for 24 h, and the results were assessed by observing the inhibition of visible growth [35,36]. The combinations of the substances were analyzed by calculating the FIC index (FICI) as follows: FIC = (MICa of the combination/MICa alone) + (MICb of the combination/MICb alone). The FIC was interpreted as: (i) a

A B
C D E F synergistic effect when ≤0.5; (ii) an additive or indifferent effect when >0.5 and ≤4; and (iii) an antagonistic effect when >4 [37]. The combination of the two components is shown graphically by a Cartesian diagram, by applying the isobole method. The non-interaction of the two components results in a straight line, whereas the occurrence of an interaction is shown by a concave isobole [38].

Conclusions
The results of this study of the antimicrobial properties of fractions and mainly of pure substances, together with their synergistic activity, is promising from the standpoint of medicinal chemistry, in the search for bioactive compounds from plants that may provide prototype molecules for the synthesis of more potent, selective, less-toxic and low-cost analogues. However we need to carry out further research to elucidate the mechanism of action of synergistic components, as well as their applications as viable antimicrobial agents.