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Enhancing activity of antibiotics against Staphylococcus aureus: Zanthoxylum capense constituents and derivatives.

ABSTRACT

Six compounds (1-6), isolated from the methanol extract of the roots of the African medicinal plant Zanthoxylum capense Thunb. (Rutaceae), and seven ester derivatives (7-13) were evaluated for their antibacterial activities and modulatory effects on the MIC of antibiotics (erythromycin, oxacillin, and tetracycline) and ethidium bromide (EtBr) against a Staphylococcus aureus reference strain (ATCC 6538). Using the same model, compounds 1-13 were also assessed for their potential as efflux pump inhibitors by a fluorometric assay that measures the accumulation of the broad range efflux pump substrate EtBr. Compounds 8 and 11 were further evaluated for their antibacterial, modulatory and EtBr accumulation effects against four additional S. aureus strains, which included two clinical methicillin-resistant S. aureus (MRSA) strains. Compounds (1-13) have not shown antibacterial activity at the concentration ranges tested. When evaluated against S. aureus ATCC 6538, oxychelerythrine (1) a benzophenanthridine alkaloid, showed the highest modulatory activity enhancing the susceptibility of this strain to all the tested antibiotics from two to four-fold. Ailanthoidiol diacetate (8) and ailanthoidiol di-2-ethylbutanoate (11) were also good modulators when combined with EtBr, increasing the bacteria susceptibility by four and two-fold, respectively. In the EtBr accumulation assay, using ATCC 6538 strain, the phenylpropanoid (+)-ailanthoidiol (6) and most of its ester derivatives (8-11) exhibited higher activity than the positive control verapamil. The highest effects were found for compounds 8 and 11 that also increased the accumulation of EtBr, using S. aureus ATCC 25923 as model. Furthermore, both compounds (8,11) were able to enhance the ciprofloxacin activity against the MRSA clinical strains tested, causing a reduction of the antibiotic MIC values from two to four-fold. The EtBr accumulation assay revealed that this modulation activity was not due to an inhibition of efflux pumps mechanism.

These results suggested that Z. capense constituents may be valuable as leads for restoring antibiotic activity against MRSA strains.

Keywords:

Bacterial resistance

Phenylpropanoids

Benzophenanthridine alkaloids

Staphylococcus aureus

Zanthoxylum capense

Introduction

The increase of bacterial resistance highlights the urgency of searching for new drugs with new action modes to treat bacterial infections. Resistance to antibiotics in bacteria can be attributed to various mechanisms including antibiotic inactivation, target-based mutation compromising drug binding, reduced permeability and efflux mechanisms by membrane proteins that pump the drugs out. Efflux acts by reducing the antibiotic intracellular concentration to levels lower than those required to exert the antibiotic effect and thus can lead to treatment failure. Particularly, the latter mechanism can convey resistance to a specific class of antibiotics or to a large number of unrelated antimicrobial agents, thus able to confer a multi-drug resistance (MDR) phenotype to bacteria (Pages and Amaral 2009).

Several approaches have been developed for overcoming bacterial MDR phenotypes such as combination therapies that include association of two or more antibiotics. Alternatively, the association of an antibiotic with bacterial resistance-modifying agents able to increase the activity of the antibiotic has also been considered a promising strategy (Worthington and Melander 2013). These compounds can act as regulators of efflux systems namely as efflux pump inhibitors (EPI) that block the activity of drug efflux pumps (Pages and Amaral 2009).

The great chemical diversity in plant-derived compounds makes them a potential source of resistance-modifying agents. The alkaloids reserpine and berberine are examples of plant-derived efflux pump inhibitors from plants; they were able to potentiate the activity of fluoroquinolones and tetracyclines against resistant bacteria (Gibbons et al. 2007; Abreu et al. 2012).

Staphylococcus aureus is a major pathogen in both hospital and the community, responsible for a wide range of infections from uncomplicated skin and soft tissue infections to more serious illnesses like pneumonia, endocarditis, and sepsis (Edelsberg et al. 2014). Particularly worrisome are multi-drug resistant strains, such as methicillin-resistant S. aureus (MRSA) that have generated a pressing need for improved MRSA therapies. In S. aureus, efflux mechanisms have been demonstrated as able to confer resistance to several antimicrobial agents, including ciprofloxacin, erythromycin, tetracyclines and biocides and thus associated with multi-drug resistance (Gibbons et al. 2007; Costa et al. 2013). The use of bacterial resistance modifiers such as EPIs could facilitate the re-introduction of therapeutically ineffective antibiotics back into clinical use such as ciprofloxacin, and erythromycin.

Zanthoxylum capense Thunb. (Rutaceae) has been widely used in traditional medicine in Africa to treat various diseases, such as colds, flu and tuberculosis (Steyn et al. 1998). In our study for identifying bioactive plant-derived compounds from African medicinal plants against infectious diseases (Madureira et al. 2012; Luo et al. 2011; Ramalhete et al. 2010, 2011, 2014), a phytochemical investigation of the constituents of the methanolic extract of Z. capense has been carried out. Bioassay-guided fractionation of the methanol extract of the roots of this plant led to the isolation of several compounds, mainly benzophenanthridine alkaloids (Luo et al. 2012, 2013). Decarine, a benzophenanthridine-type alkaloid, has shown a significant antimycobacterial activity against Mycobacterium tuberculosis in vitro, and ex vivo within human macrophages (Luo et al. 2013) and some compounds, such as 6-acetonyldihydronitidine, showed a promising antibacterial activity against Staphylococcus aureus (Luo et al., 2012).

Herein, we describe the isolation and identification of six compounds (1-6) from the same plant. Furthermore, the aromatic amide (+)-tembamide (5), and the phenylpropanoid (+)-ailanthoidiol (6) were esterified with different acylating reagents to afford seven alkanoyi/aroyl esters (7-13). Compounds 1-13 were evaluated for their antibacterial activity against S. aureus, including MRSA and methicillin-susceptible S. aureus (MSSA) strains. The compounds were also assessed for their potential activity as antibiotic modulators and efflux pump inhibitors. In order to evaluate the modulatory effects were selected fluorquinolones (ciprofloxacin), macrolides (erythromycin) and tetracyclines, which are important antibiotics to treat infections caused by S. aureus MDR. Multiresistant strains often over-express efflux pump genes that may correlate to multiresistant phenotypes (Costa et al. 2013). Thus, it would be important to restore the effectiveness of these antibiotics.

Materials and methods

General

Optical rotations were obtained using a Perkin-Elmer 241 polarimeter. IR spectra were determined on a Shimadzu IRAffinity-1 spectrophotometer. NMR spectra were recorded on a Bruker ARX-400 NMR spectrometer ([sup.1]H 400 MHz, [sup.13]C 100.61 MHz), using CD[Cl.sub.3] and MeOD as solvents. ESIMS spectra were taken on a Micromass[R] Quattro micro[TM] API. Column chromatography (CC) was carried out using Merck silica gel 60 (230-400 mesh). TLC was performed on Merck silica gel 60 F254 plates and visualized under UV light and by spraying with sulfuric acid-MeOH (1:1) followed by heating. Preparative TLC was performed with silica plates 20 x 20 cm, 0.5 mm thick (Merk, ref. 1.05744), with visualization under UV light. HPLC was carried out on a Merck-Hitachi instrument, with a UV Hitachi L-2400 detector, using a Merck LiChrospher 100 RP-18 (5 [micro]m, 125 x 4 mm) column. Preparative HPLC was performed on a modular LaPrep system, La prep P100 pump, UV La prep P314 detector and a Merck Lichrospher 100 RP-18 column (10 [micro]m, 250 x 10 mm).

Plant material

The roots of Zanthoxylum capense were collected in southern Mozambique (Machava and Massingir) and were identified by the botanist Dr. Silva Mulhovo. A voucher specimen (No. 45/SM) was deposited at the herbarium (LMA) of the Instituto de Investigacao Agraria de Mozambique (1IAM), Maputo, Mozambique.

Tested compounds

The chemical structures of compounds (1-13) are presented in Fig. 1. These included oxychelerythrine (1), oxynitidine (2), arnottianamide (3), ([+ or -])-syringaresinol (4), (+)-tembamide (5), (+)-ailanthoidiol (6), (+)-tembamide acetate (7), (+)-ailanthoidiol diacetate (8), (+)-ailanthoidiol dibutanoate (9), (+)-ailanthoidiol di-2-methylbutanoate (10), (+)-ailanthoidiol di-2-ethylbutanoate (11), (+)-ailanthoidiol didodecanoate (12), and (+)-ailanthoidiol dibenzoate (13). The isolation of compounds 1-6 and the preparation of compounds 7-13 are described below. The purity of all the compounds was more than 95 % based on HPLC analysis and NMR spectroscopy.

Extraction and isolation

The air-dried powdered roots (4.3 kg) of Z capense were exhaustively extracted with methanol (10 x 8 1) as previously described (Luo et al. 2012). Briefly, the MeOH residue (969 g) was suspended in a solution of MeOH/[H.sub.2]0 (9:1) (5 1) and partitioned with n-hexane (3 x 51) to yield the n-hexane extract (110 g). The aqueous MeOH layer was further diluted into 50 % MeOH solution and extracted with C[H.sub.2][Cl.sub.2] (3 x 91) to obtain the C[H.sub.2][Cl.sub.2] extract (100 g), which was chromatographed over a silica gel column (8 x 120 cm, 1.6 kg Si[0.sub.2]) eluted with mixtures of n-hexane-EtOAc (1:0 to 0:1) and EtOAc-MeOH (1:0 to 9:1), to yield 16 crude fractions (FD1-16). Fraction FD8 (2.71 g) was chromatographed, eluting with mixtures of n-hexane/C[H.sub.2][Cl.sub.2]/MeOH (1:4:0 to 0:0:1) to give eight fractions (FD8.1-FD8.8). Fraction FD8.7 (1.08 g) was re-chromatographed using C[H.sub.2][Cl.sub.2]/MeOH (1:0 to 99:1) to yield 14 subfractions (FD8.7.1-FD8.7.14). Subfraction FD8.7.3 was purified by semi-preparative RP-HPLC (254 nm, MeOH/[H.sub.2]O, 13:7, 4 ml/min) to afford compound 1 (3 mg). Recrystallization of subfractions FD8.7.4 and FD8.7.12 from EtOAc/n-hexane yielded compounds 5 (51 mg) and 6 (56 mg), respectively. Similarly, chromatography of fraction FD8.8 (420 mg), using C[H.sub.2][Cl.sub.2]/MeOH (1:0 to 99:1), gave rise to nine subfractions (FD8.8.1-FD8.8.9). Subfraction FD8.8.1 was recystallized from EtOAc/MeOH to give compound 2 (23 mg), and recrystallization of subfractions FD8.8.5 and FD8.8.8, using n-hexane/EtOAc, afforded compounds 3 (22 mg) and 6 (120 mg), respectively. Fraction FD11 (6.17 g) was subjected to a silica gel column, using C[H.sub.2][Cl.sub.2]/MeOH (99.6:0.4 to 9:1) as eluents yielding 9 fractions (FD11.1-FD11.9). From fraction FD11.1, compound 4 (26 mg) was obtained by preparative thin layer chromatography (C[H.sub.2][Cl.sub.2]/MeOH 24:1, Rf 0.3) followed by HPLC purification (254 nm, MeOH/[H.sub.2]O13:7,4 ml/min).

Preparation of ester derivatives

General preparation method

Compound 5/6 (1 eq.) was dissolved in dry pyridine (1 ml) at room temperature under [N.sub.2]. Afterward, acetic anhydride or suitable chloride (8 eq.) was added and the solution was stirred for 6 h. After removing the excess of reagents under vacuum at 40[degrees]C, the residue was purified by column chromatography, eluting with mixtures of n-hexane/C[H.sub.2][Cl.sub.2] to obtain the ester derivatives (7-13).

(+)-Tembamide acetate (7): Obtained from reaction with acetic anhydride (Merck KGaA, Darmstradt, Germany). The residue was purified by flash column chromatography (silica gel, C[H.sub.2][Cl.sub.2]/MeOH 99.8:0.2 to 99:1) to afford 4 mg of white crystals. ESIMS, m/z: 336 [[M + Na].sup.+]. [sup.1]H NMR(400 MHz, MeOD): [delta] 7.72 (2H, d, J = 8 Hz, H-2', H-6'), [delta] 7.50 (1H, t, J = 8 Hz, H-4'), [delta] 7.43 (2H, t, J = 8 Hz, H-3', H-5'), [delta] 7.33 (2H, d, J = 8 Hz, H-2, H-6), [delta] 6.91 (2H, d, J = 8 Hz, H-3, H-5), [delta] 6.46 (1H, br t, J = 4 Hz, N[H.bar]-9), [delta] 5.4 (1H, t, J = 4 Hz, H-7), [delta] 3.84 (2H, m, H-8), [delta] 3.81 (3H, s, OC[[H.bar].sub.3]-4), [delta] 2.10 (3H, s, C7-COOC[[H.bar].sub.3]). [sup.13]C NMR (100 MHz, MeOD): [delta] 170.78 (C = 0 C7), [delta] 167.23 (C-10), [delta] 159.70 (C-4), [delta] 134.21 (C-1), [delta] 131.58 (C-1'), [delta] 129.60 (C-4'), [delta] 128.61 (C-2', C-6'), [delta] 127.92 (C-3', C-5'), [delta] 126.85 (C-3, C-5), [delta] 114.09 (C-2. C-6), [delta] 74.32 (C-7), [delta] 55.29 (4-OMe), [delta] 44.98 (C-8), [delta] 21.24 ([C.bar][H.sub.3]COO-7).

(+)-Ailanthoidiol diacetate (8): Obtained from reaction with acetic anhydride (Merck KGaA, Darmstradt, Germany). The residue was purified by flash column chromatography (silica gel, n-hexane/C[H.sub.2][Cl.sub.2]1:0 to 0:1) to afford 17 mg of a colorless oil, 1R (film) Vmax 1737,1732,1606,1512,1379,1363,1238,1176,1026,970 [cm.sup.-1]. ESIMS m/z: 343 [[M + Na].sup.+]. [sup.1]H NMR (400 MHz, CD[Cl.sub.3]): [delta] (ppm) 7.32 (2H, dd, J = 2,8 Hz, H-2, H-6), 6.84 (2H, dd. J = 2,8 Hz, H-3, H-5), 6.59 (1H, br d, J= 15.6 Hz, H-7), 6.15 (1H, dt, J = 15.6,4.4 Hz, H-8), 4.70 (2H, d, J = 1.2,4.4 Hz, H-9), 4.03 (2H, m, H-l'), 3.98 (2H, d, J = 6 Hz, H-4'), 2.10 (1H, m, H-3'), 2.09 (3H, s, H-2'"), 2.05 (3H, s, H-2"), 1.89 (1H, m, H-2'b), 1.65 (1H, m, H-2'), 1.01 (3H, d,J = 6.8 Hz, CH3-3'). [sup.13]C NMR (100 MHz, CD[Cl.sub.3]): [delta] (ppm) 171.23 (C-1"), 170.94 (C - 1'"), 158.91 (C-4), 134.06 (C7), 128.92 (C-1), 127.87 (C-2, C-6), 120.80 (C-8), 114.54 (C-3, C-5), 69.06 (C-4'), 65.66 (C-l'), 65.36 (C9), 32.80 (C-3'), 29.76 (C-2'), 21.08 (C - 2", C - 2'"), 16.84 ([C.bar][H.sub.3]-3').

(+)-Ailanthoidiol dibutanoate (9): Obtained from reaction with butanoyl chloride. The residue was purified by flash column chromatography (silica gel, n-hexane/C[H.sub.2][Cl.sub.2],1:0 to 0:1) to afford 10 mg of a colorless oil. IR(film) [v.sub.max] 1737, 1732, 1606, 1512, 1247, 1174, 966 [cm.sup.-1]. ESIMS, m/z: 399 [[M + Na].sup.+]. [sup.1]H RMN (400 MHz, CD[Cl.sub.3]): [delta] (ppm) 7.31 (2H, dd, J = 2,8 Hz, H-2, H-6), 6.83 (2H, dd, J = 2,8 Hz, H-3, H-5), 6.58 (1H, brd, J = 15.6 Hz, H-7), 6.13 (1H, dt, J = 15.6, 4.4 Hz, H-8), 4.70 (2H, dd, J = 1.2,4.4 Hz, H-9), 4.02 (2H, m, H-l'), 3.99 (2H, d, J = 5.6 Hz, H-4'), 2.30 (4H, dt, J = 16,7.6 Hz, H-2", H - 2'"), 2.09 (1H, m, H-3'), 1.89 (1H, m, H-2"b), 1.66 (5H, m, H-2'a, H-3", H - 3'"), 1.01 (3H, d, J = 6.8 Hz, C[[H.bar].sub.3]-3'), 0.95 (6H, t, J = 7.6 Hz, H-4", H - 4'"). [sup.13]C RMN (100 MHz, CD[Cl.sub.3]): 8 (ppm) 173.74 (C-1"), 173.48 (C - 1"'), 158.84 (C-4), 133.84 (C-7), 129.14 (C-1), 127.81 (C-2, C-6), 120.94 (C-8), 114.49 (C-3, C-5), 68.74 (C-4'), 65.63 (C-1'), 65.09 (C-9), 36.20 (C-2", C - 2'"), 32.79 (C-3'), 29.78 (C-2'), 18.42 (C-3", C - 3'"), 16.82 (CH3-3'), 13.67 (C-4", C - 4'").

(+)-Ailanthoidiol di-2-methylbutanoate (10): Obtained from reaction with 3-methylbutanoyl chloride. The residue was purified by flash column chromatography (silica gel, n-hexane/C[H.sub.2][Cl.sub.2],100:0 to 0:100) to afford 10 mg of a colorless oil. IR (film) [v.sub.max] 2966, 2933, 2877, 1737, 1732, 1606, 1512, 1462, 1246, 1176, 1149, 1014, 968 [cm.sup.-1]. ESIMS, m/z: 427 [[M + Na].sup.+]. [sup.1]H RMN (400 MHz, CD[Cl.sub.3]): [delta] (ppm) 7.31 (2H, dd, J = 2, 8 Hz, H-2, H-6), 6.83 (2H, dd, J = 2, 8 Hz, H-3, H-5), 6.58 (1H, br d, J = 15.6 Hz, H-7), 6.13 (1H, dt, J = 15.6,4.4 Hz, H-8), 4.70 (2H, dd, J = 1.2,4.4 Hz, H-9), 4.02 (2H, m, H-1'), 3.99 (2H, d, J = 5.6 Hz, H-4'), 2.39 (2H, m, H-2"b, H - 2'"), 2.08 (1H, m, H-2'a), 1.91 (1H, m, H2'b), 1.68 (2H, m, H-3", H - 3'"), 1.64 (1H, m, H-3'), 1.49 (2H, m, H-2"a, H - 2"'a), 1.15 (6H, t, J = 7.2 Hz, H-4", H - 4'"), 1.01 (3H, d,J = 6.8 Hz, CH3-3'), 0.91 (3H, t, J = 7.2 Hz, H-4"/H - 4'"), 0.90 (3H, tj = 7.2 Hz, H4"/H - 4'"). ,3C RMN (100 MHz, CD[Cl.sub.3]): [delta] (ppm) 176.80(C-1"), 176.61 (C-1'"), 158.87 (C-4), 133.76 (C-7), 129.02 (C-1), 127.85 (C-2, C-6), 121.11 (C-8), 114.54 (C-3, C-5), 68.69 (C-4'), 65.68 (C-1'), 65.07 (C-9), 41.12 (C-2", C - 2'"), 32.82 (C-3'), 29.88 (C-2'), 26.65 (C-3", C - 3'"), 16.83 ([C.bar][H.sub.3]-3'), 16.62 (C-4"/C - 4'"), 11.68 (C-4"/C - 4'").

(+)-Ailanthoidiol di-2-ethylbutanoate (11): Obtained from reaction with 2-ethylbutanoyl chloride. The residue was purified by flash column chromatography (silica gel, n-hexane/C[H.sub.2][Cl.sub.2],1:0 to 0:1) to afford 10 mg of a colorless oil. IR (film) vmax 1732, 1606, 1512, 1462, 1267, 1247, 1174, 1145, 964 [cm.sup.-1]. ESIMS, m/z: 455 [[M + Na].sup.+]. [sup.1]H RMN (400 MHz, CD[Cl.sub.3]): [delta] (ppm) 7.31 (2H, dd, J = 2,8 Hz, H-2, H-6), 6.83 (2H, dd, J = 2,8 Hz, H-3, H-5), 6.58 (1H, br d, J = 15.6 Hz, H-7), 6.13 (1H, dt, J = 15.6,4.4 Hz, H-8), 4.72 (2H, d, J = 6.4 Hz, H-9), 4.02 (2H, m, H-1'), 4.00 (2H, d, J = 5.6 Hz, H-4'), 2.22 (2H, m, H-2", H - 2'"), 2.10(1H, m, H-2'b), 1.92 (1H, m, H-2'a), 1.60 (5H, m, H-3',H-3"a, H - 3"'a) 1.53 (4H, m, H-3"b, H - 3"'b), 1.02 (3H, d, J = 6.8 Hz, C[[H.bar].sub.3]-3'), 0.90 (12H, m, H-4", H - 4'"). 13C RMN (100 MHz, CD[Cl.sub.3]): [delta] (ppm) 176.34 (C-1"), 176.13 (C-1'"), 158.87 (C-4), 133.77 (C-7), 129.04 (C-1), 127.83 (C-2, C-6), 121.16 (C-8), 114.54 (C-3, C-5), 68.61 (C-4'), 65.68 (C-l'), 64.94 (C-9), 48.97 (C-2", C - 2'"), 32.84 (C-3'), 29.87 (C-2'), 25.06 (C3", C - 3'"), 16.88 (C[H.sub.3]-3'), 11.88 (C-4", C - 4'").

(+)-Ailanthoidiol didodecanoate (12): Obtained from reaction with dodecanoyl chloride. The residue was purified by flash column chromatography (silica gel, n-hexane/C[H.sub.2][Cl.sub.2], 1:0 to 0:1) to afford 14 mg of a colorless oil. IR(film) [v.sub.max] 1737,1606,1512,1465,1238, 1174, 968 [cm.sup.-1]. ESIMS, m/z: 623 [[M + Na].sup.+]. [sup.1]H RMN (400 MHz, CD[Cl.sub.3]): 8 7.31 (2H, dd, J = 2,8 Hz, H-2, H-6), 6.83 (2H, dd, J = 2,8 Hz, H-3, H-5), 6.58 (1H, brd, J = 15.6 Hz, H-7), 6.13 (1H, dt, J = 15.6,4.4 Hz, H-8), 8 4.70 (2H, d, J = 6.4 Hz, H-9), 8 4.01 (2H, m, H-1'), 3.98 (2H, d, J = 5.6 Hz, H-4'), 2.31 (4H, dt, J = 7.2,15.6 Hz, H-2", H - 2'"), 2.08 (1H, m, H-3'), 1.90 (1H, m, H-2'b), 1.63 (5H, m, H-2'a, H-3", H - 3'"), 1.25 (32H, m, H-4" to H-11", H - 4'" to H-l 1'"), 1.01 (3H, d, J = 6.8 Hz, C[H.sub.3]-3'), 0.87 (6H, t, J = 6.8 Hz, H-12", H - 12"').13C RMN (100 MHz, CD[Cl.sub.3]): 8 174.00 (C-1"), 173.74 (C-l'"), 158.87 (C-4), 133.89 (C-7), 130.21 (C-1), 127.85 (C-2, C-6), 120.98 (C-8), 114.52 (C-3, C-5), 68.79 (C-4'), 65.67 (C-l'), 65.16 (C-9), 34.40 (C-2", C-2'"), 31.93 (C-3'), 29.62 (C-2'), 29.15-29.46 (C-3" to C-10", C-3'" to C- 10'"), 24.96 (C-11", C11'"), 16.88 ([C.bar][H.sub.3]-3'), 14.15 (C-12", C- 12'").

(+)-Ailanthoidiol dibenzoate (13): Obtained from reaction with benzoyl chloride. The residue was purified by flash column chromatography (silica gel, n-hexane/C[H.sub.2][Cl.sub.2], 1:0 to 0:1) to afford 9 mg of a colorless oil. IR(film) [v.sub.max] 1720, 1714, 1604, 1510, 1450, 1271, 1249, 1174, 1111, 1070, 1026, 968, 709 [cm.sup.-1]. ESIMS, m/z: 467 [[M + Na].sup.+]. [sup.1]H RMN (400 MHz, CD[Cl.sub.3]): [delta] (ppm) 8.08 (2H, d, J = 7.2 Hz, H-3", H-7"), 8.03 (2H, d, J = 8.4 Hz, H - 3'", H - 7'"), 7.56 (2H, m, H-5", H - 5'"), 7.44 (4H, m, H-4", H-6", H - 4'", H - 6'"), 7.34 (2H, dd. J = 2, 8 Hz, H-2, H-6), 6.86 (2H, dd.J = 2, 8 Hz, H-3, H-5), 6.70 (1H, br d, J = 15.6 Hz, H-7), 6.28 (1H, dt,J = 15.6,4.4 Hz, H-8), 4.96 (2H, d, J = 1.2, 4.4 Hz, H-9), 4.25 (2H, d,J = 6 Hz, H-4'), 4.08 (2H,m, H-1'), 2.26 (1H, m, H-3'), 2.00 (1H, m, H-2'b), 1.76 (1H, m, H-2'a), 1.11 (3H, d,] = 6.8 Hz, CU3-3'). 13C RMN (100 MHz, CD[Cl.sub.3]): 5 (ppm) 166.87 (C-1"), 166.48 (C - 1'"), 158.90 (C-4), 134.13 (C-5", C - 5'"), 132.98 (C-7), 130.29 (C1), 129.55 (C-4", C - 4'" e C-6", C - 6'"), 128.96 (C-2", C - 2'"), 128.39 (C-3", C - 3'" e C-7", C - 7'"), 127.92 (C-2, C-6), 120.90 (C-8), 114.59 (C-3, C-5), 69.47 (C-4'), 65.87 (C-1'), 65.68 (C-9), 32.94 (C-3'), 29.99 (C-2'), 17.02 (C-3').

Bacterial strains and chemicals

S. aureus ATCC 6538, a reference strain, was used in the preliminary screening for antibacterial activity and antibiotic modulatory activity of the compounds in study. In further assays, four additional S. aureus strains were tested, which included the reference strain ATCC 25923, its derivate, adapted to 50 mg/1 of ethidium bromide (EtBr), ATCC 25923EtBr and overexpressing the norA efflux pump gene (Couto et al. 2008) and two clinical MRSA strains with increased efflux activity, S. aureus SMI, a methicillin and ciprofloxacin-resistant strain (Costa et al. 2011) and S. aureus SM39, methicillin resistant with reduced susceptibility to biocides and carrying the qacA gene (Costa 2013). All strains were grown at 37 [degrees]C in tryptic soy broth (TSB, Oxoid, Basingstoke, UK) or tryptic soy agar (TSA) plates, for 18 h, except strain [ATCC25923.sub.EtBr], which was grown in TSB/TSA supplemented with 50 mg/1 of EtBr. Antibiotics and EtBr were purchased from different sources, as follows: erythromycin, oxacillin, tetracycline and EtBr from Sigma-Aldrich (Madrid, Spain) and ciprofloxacin from Fluka Chemie GmbH (Buchs, Switzerland). Verapamil, used as positive control in the accumulation assay, was acquired from Sigma-Aldrich.

Minimum inhibitory concentration (MIC) determination

Minimum inhibitory concentration values were determined using the two-fold broth microdilution method, in Mueller-Hinton broth (MHB, Oxoid), following the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2013). The MIC, defined as the lowest concentration of compound at which no visible growth (turbidity) was detected, was determined after 18 h of incubation at 37[degrees]C, except for oxacillin, for which results were evaluated after 24 h incubation.

The compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and tested at concentrations ranging from 1.6 to 100 [micro]g/ml. Due to a solubility issue, compounds 2 and 3 were tested at the concentration range of 0.8-50 [micro]g/ml. Erythromycin, oxacillin and tetracycline were tested against S. aureus ATCC 6538 at concentrations ranging from 0.05 to 10 [micro]g/ml. Ciprofloxacin was tested at concentrations ranging from 2 to 1024 [micro]g/ml for S. aureus SMI, and 0.015 to 8 [micro]g/ml for the other strains. EtBr was tested ranging from 0.125 to 64 [micro]g/ml against S. aureus ATCC 6538 and SMI strains, 0.05 to 25 [micro]g/ml against S. aureus ATCC 25923 and 0.78 to 400 [micro]g/ml for S. aureus ATCC 25923EtBr and SM39 strains. Verapamil was tested at concentrations ranging from 25 to 1600 [micro]g/ml.

Combination effect of compounds with antibiotics

The modulatory effect of the compounds was evaluated by determination of MIC values of the antibiotics and EtBr in the presence of sub-inhibitory concentrations of the compounds, 100 [micro]g/ml or 50 [micro]g/ml (2 and 3). The MICs were carried out by the two-fold broth microdilution method, using a serial dilution of the antibiotics ranging from 0.025 to 1.6 [micro]g/ml, and 0.5 to 32 [micro]g/ml for EtBr.

The modulation factor (MF) was used to evaluate the modulating effects of compounds on MICs of antibiotics according to the following formula: MF = MIC antibiotic/MIC antibiotic + modulator. The fractional inhibitory concentration (FIC) of the antibiotics was also calculated according to the following formula: FIC = MIC of antibiotic in combination/MIC of antibiotic alone.

Real-time EtBr accumulation assay

In order to evaluate if the compounds behaved as efflux pump inhibitors, an EtBr accumulation assay was carried out by a real-time fluorometric method using a Rotor-Gene 3000(tm) (Corbett Research, Sydney, Australia) (Viveiros et al. 2010). In these assays, EtBr is used as a fluorochrome, exploring its characteristics as a broad range efflux pump substrate. EtBr is capable to emit weak fluorescence in aqueous solutions (outside cell) and higher fluorescence inside the cell, allowing the detection of its accumulation inside the cell: since accumulation of EtBr and its extrusion from bacteria are the result of the balance between EtBr entry by passive diffusion (influx) and the extrusion activity of efflux pump systems (Viveiros et al. 2010). The experiments were carried out with EtBr concentrations that did not affect cell viability, below 'A the MIC value for EtBr.

S. aureus cultures were grown in TSB at 37 [degrees]C with shaking until they reach an optical density of 0.6 at 600 nm ([OD.sub.600] nm). The cells were collected by centrifugation at 13,000 rpm for 3 min and the pellet washed twice with a Phosphate Buffered Saline (PBS) solution. A cellular suspension was prepared in PBS with an [OD.sub.600] nm adjusted to 0.6. Aliquots of 50 [micro]l of the bacterial suspension (final [OD.sub.600] nm of 0.3) were added to 0.2 ml microtubes containing: (i) 50 [micro]l of PBS; (ii) 50 [micro]l of 2X the most appropriate EtBr concentration (0.25 [micro]g/ml for strains ATCC 6538 and ATCC 25923 and 0.5 [micro]g/ml for ATCC 25923EtBr, SMI and SM39); (iii) 50 [micro]l of a solution of the compound to be tested at 100 [micro]g/ml in PBS (final concentration of 50 [micro]g/ml) plus 2X the most appropriate EtBr concentration; (iv) 50 [micro]l of a solution of verapamil, a known efflux inhibitor which was used as a control (Costa et al. 2011), at 400 [micro]g/ml in PBS (final concentration of 200 [micro]g/ml) plus 2X the most appropriate EtBr concentration. EtBr fluorescence data was acquired (530/585 nm) every minute for 60 min at 37[degrees]C. The relative final fluorescence (RFF) (Machado et al. 2011) was determined to compare the inhibitory activity of the compounds which is calculated according to the following formula: RFF = ([F.sub.compound] - [F.sub.control])/[F.sub.control] , where [F.sub.compound] corresponds to the fluorescence at 60 min of the EtBr accumulation curve in the presence of a given compound and [F.sub.control] is the fluorescence at 60 min of the EtBr accumulation curve in the absence of that compound.

Results and discussion

Isolation and molecular derivatization of compounds

The air-dried powdered roots of Z. capense were exhaustively extracted with MeOFI (Luo et al. 2012). Bioassay-guided fractionation of the methanol extract led to the isolation of several compounds, mainly isoquinoline alkaloids of the benzophenanthridine-type (Luo et al. 2012, 2013). Further fractionation of the methanol extract of the roots of Z. capense yielded six compounds namely three benzophenanthridine alkaloids, oxychelerythrine (1), oxynitidine (2) and arnottianamide (3), the lignan ([+ or -])-syringaresinol (4), the aromatic amide (+)-tembamide (5), and the phenylpropanoid (+)-ailanthoidiol (6) (Fig. 1). The structures of the compounds were elucidated on the basis of spectroscopic methods, including 2D-NMR experiments, and comparison with the data reported in the literature (Sheen et al. 1994; Yang et al. 2009; Miao et al. 2011; Hsiao and Chiang 1995; Yadav et al. 2001; Kan et al. 2011). Acylation with acetic anhydride or alkanoyl/aroyl chlorides of the free hydroxyl groups of (+)-tembamide (5) and ailanthoidiol (6), isolated in higher amounts, yielded seven new esters (7-13, Fig. 1) whose NMR data were assigned by comparing with those of the parent compounds (5,6). In this way, the main differences between the [sup.1]H NMR and [sup.13]C NMR spectra of 6 and the esters 8-13 were observed for the signals of H-4' and H-9, which showed a significant paramagnetic effect, appearing 0.4 to 0.7 ppm downfield, and the corresponding a-carbons C-4' and C-9, which were also deshielded ([DELTA][[delta].sub.C] [approximately equal to] +0.9 and + 1.2 ppm, respectively), as expected. Moreover, diamagnetic effects were also displayed at C-3' and C-8 ([DELTA][[delta].sub.C] [approximately equal to] -0.8 and 5.4 ppm, respectively, [gamma]-carbons) and C[H.sub.3]-3' and C-7 were shifted downfield ([DELTA][[delta].sub.c] [approximately equal to] +0.1 and +2.8 ppm, respectively, [gamma]-carbons). Similarly, in compound 7, the acetylated derivative of (+)-tembamide (5), the signal of H-7 was observed downfield ([DELTA][[delta].sub.H] + 1.05) and paramagnetic effects were observed at C-7 ([DELTA][[delta].sub.c] = + 2.5), C-2/C-6 ([DELTA][[delta].sub.C] = +0.76) and C-8 and C-1 appeared upfield ([DELTA][[delta].sub.C] = -2.29 and -0.3, respectively).

Preliminary screening of compounds: combination and accumulation assays

Combination effect of compounds with antibiotics

In a first approach, the in vitro antibacterial activity of the compounds (1-13) was tested against S. aureus ATCC 6538; no compound showed antibacterial activity at the concentration ranges tested: 1.6-100 [micro]g/ml for 1, 4-13 and 0.8-50 [micro]g/ml for 2 and 3. In order to search for antibiotic modulatory activity, a combination assay between compounds 1-13 and the antibiotics erythromycin, tetracycline and oxacillin and EtBr was performed. Modulation factors (MF) were determined to express and compare the effect displayed by the compounds on the antibiotics MIC reduction. The fractional inhibitory concentration of the antibiotics (FIC) was also calculated (Lechner et al. 2008).

As shown in Table 1, the benzophenanthridine alkaloid oxychelerythrine (1) exhibited the best modulation effect on the antibiotics and on EtBr. The MICs of oxacillin for S. aureus decreased four-fold (MF = 4; FIC = 0.25) and the MICs of erythromycin, tetracycline and EtBr two-fold (MF = 2; FIC = 0.5) after adding oxychelerythrine (1). The other two benzophenanthridine alkaloids oxynitidine (2) and arnottianamide (3) also showed modulatory effect in combination with tetracycline, by decreasing the MIC value forS. aureus two-fold (MF = 2; FIC = 0.5). The remaining natural compounds (4-6) showed no modulatory effect in combination with the antibiotics and EtBr against the S. aureus ATCC 6538. Nevertheless, tembamide (5), and ailanthoidiol (6) afforded chemical modifications that gave rise to compounds with improved activity. Indeed, among the ester derivatives of ailanthoidiol (6), compounds 8, 10 and 11, also displayed modulation effect on EtBr, with MF values of 4 (FIC = 0.25, compound 8) and 2 (FIC = 0.5, compounds 10,11).

Besides depending on structural features, the modulating activity of compounds also depends on their physicochemical properties namely the lipophilic character. Among the isolated compounds (1-6) the higher lipophilicity of the three isoquinoline alkaloids oxychelerythrine (1), oxynitidine (2), and arnottianamide (3) (log P = 3.93, 3.74 and 3.50, respectively; Fig. 1) might be important for their modulating effect. It is interesting to note that the three compounds (1-3) belong to the group of isoquinoline alkaloids as berberine. The latter is a plant lipophilic alkaloid that is able to modify the activity of antibiotics by increasing membrane permeability (Abreu et al. 2012), suggesting that compounds 1-3 may also interact similarly with the membrane. Concerning the active ailanthoidiol (6) acyl derivatives (8, 10,11) their higher lipophilicity (log P = 3.88, 6.71, 7.72, respectively; Fig. 1) when compared with that of the parental compound (6, log P = 2.47), might also explain, partially, their antibiotics potentiation.

Real-time EtBr accumulation assay

A relevant resistance mechanism in S. aureus is efflux by protein transporters (Li and Nikaido 2004). Therefore, in order to search for compounds that can be used in combination with antibacterial agents and thus restore their effectiveness by inhibiting their extrusion, the potential activity of compounds 1-13 as efflux pump inhibitors was evaluated using a real-time fluorometric method (Viveiros et al. 2010) and S. aureus ATCC 6538 as a model (Fig. 2A). The capacity of each compound to promote an increase in EtBr accumulation, indicative of a potential efflux pump inhibitory activity, was evaluated and compared to the activity presented by verapamil, a known efflux pump inhibitor in S. aureus. The effect of the compounds exerted on EtBr accumulation in S. aureus ATCC 6538 was compared by determining the relative final fluorescence values (RFF) for each compound and verapamil (Table 2). As can be observed in Table 2, ailanthoidiol (6) along with most of its ester derivatives (8-11) exhibited higher RFF values than the positive control verapamil (RFF 0.55). Compounds 11 and 8 were the most effective in increasing the EtBr accumulation with RFF values of 2.84 and 1.47, respectively, emphasizing the importance of the acyl moiety for the activity when compared with that of the parental compound (6). In addition, the two benzophenanthridine alkaloids, oxynitidine (2) and oxychelerythrine (1) also promoted a moderate increase of EtBr accumulation in S. aureus cells, which was comparable to that of verapamil (RFF values of 0.79 and 0.76, respectively). No significant enhancement of EtBr accumulation was observed in the presence of compounds 3-5,7,12 and 13.

Further combination and accumulation assays of compounds 8 and 11

The joint analysis of the data gathered by MIC determination in combination with the compounds and real-time fluorometry showed that compounds ailanthoidiol diacetate (8) and ailanthoidiol di-2ethylbutanoate (11) exhibited a modulating effect in combination with EtBr and a good capacity to enhance EtBr accumulation in S. aureus cells, suggesting that they could behave as efflux pump inhibitors. Therefore, compounds 8 and 11 were selected for further assays against additional antibiotic susceptible and resistant S. aureus strains. The activity of both compounds was evaluated in combination with the fluoroquinolone ciprofloxacin (substrate of the S. aureus efflux pumps, including NorA) and EtBr against two reference strains S. aureus ATCC 6538 and ATCC 25923, and three strains for which increased efflux activity has been detected and associated with resistance to antibiotics, EtBr or biocides, namely: S. aureus ATCC [25923.sub.EtBr], a norA-overexpressing strain (Couto et al. 2008), S. aureus SM39 harbouring the biocide efflux pump gene qacA (Costa 2013) and the ciprofloxacin-resistant strain S. aureus SMI (Costa et al. 2011) (Table 3). Both compounds (8,11) showed a modulation effect in combination with ciprofloxacin against all S. aureus strains, except for the ciprofloxacin-resistant strain SMI. However, in combination with EtBr, compound 8 showed a higher effect than compound 11, with MF values of 8 (FIC = 0.125) forS. aureus SM39 and ATCC 25923EtBr and MF values of 4 (FIC = 0.25) forS. aureus SMI and the two reference strains.

The potentiation of EtBr antibacterial activity in all strains further supports that ailanthoidiol diacetate (8) could be a potential inhibitor of efflux pumps in S. aureus. To test this hypothesis, EtBr accumulation assays were performed to evaluate compounds 8 and 11 as efflux pump inhibitors against the same set of strains. The data gathered revealed that the two compounds showed a mild effect on EtBr accumulation in the reference strain ATCC 25923 (Fig. 2B) and that neither compound was able to promote an increase in EtBr accumulation in the strains that present increased efflux activity, ATCC 25923EtBr. SMI and SM39 (Table 4). Despite the observed modulatory effects in combination with ciprofloxacin and EtBr (Table 3) compounds 8 and 11 exhibited a weak efflux pump inhibitory activity. The results suggest that although the compounds are capable of some level of inhibition of the intrinsic activity of S. aureus efflux pumps, they are unable to surpass increased efflux activity, as the one present in the clinical isolates and laboratory-derived strains studied. Thus, taken together these results suggest that the weak efflux pump inhibitory activity of compounds 8 and 11 may only partially justify their potentiation effect of ciprofloxacin and EtBr against S. aureus resistant strains and other mechanism should also contribute for their activity. The potentiation of antibiotics activity by lipophilic resistance-modifying agents through the increase of membrane permeability of bacterial cells, mainly due to the perturbation of the lipid fraction of membrane, has been considered an important mechanism (Abreu et al. 2012). This mode of modulation due to membrane-disturbing properties has been reported for phenolic compounds with structures similar to these of compounds 8 and 11 (Abreu et al. 2012).

Conclusion

Summarizing, Z. capense benzophenanthridine alkaloids and some ailanthoidiol acyl derivatives have shown significant antibiotic modulatory effects against S. aureus strains and may be valuable as leads for fighting bacterial resistance to antibiotics. A common feature of these compounds is lipophilicity, suggesting that this is a key factor for modulating antibiotic resistance in S. aureus strains. Although ailanthoidiol derivatives have not manifested an efflux inhibition effect in S. aureus strains with increased efflux activity, they revealed to be good resistance modulators against these strains and may act by other modulation mechanisms, such as interaction with membranes, suggesting their ability as synergistic agents in combinatory therapies, restoring the antibiotic activity against S. aureus strains.

ARTICLE INFO

Article history:

Received 6 September 2014

Revised 12 January 2015

Accepted 10 February 2015

Conflict of interest

The authors declare that there are no known conflicts of interest associated with this publication.

Acknowledgments

This study was supported by the Portuguese Foundation for Science and Technology (FCT) (projects: PTDC/QEQ-IVIED/0905/2012, PEst-OE/SAU/UI4013/2011, REDE/1518/REM2005, grant numbers: BPD/37179/2007 and PTDC/BIA-MIC/105509/2008). SSC was supported by FCT grant BPD/97508/2013. The authors thank Dr. Patricia Gaspar, from the Portuguese Embassy in Mozambique, as well as the Portuguese Office of International Affairs for plant transport.

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Vanessa Cabral (a), Xuan Luo (a), Elisabete Junqueira (b), Sofia S. Costa (b,c), Silva Mulhovo (d), Aida Duarte (a), Isabel Couto (b,c), Miguel Viveiros (b,e), Maria-Jose U. Ferreira (a), *

(a) Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa. Av. Professor Cama nnto, 1649-003 Lisboa, Portugal

(b) Grupo de Micobacterias, Unidade de Microbiologia Medica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal

(c) Centro de Recursos Microbiologicos (CREM), Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal

(d) Centro de Estudos Mogambicanos e de Etnociencias (CEMEC), Faculdade de Ciencias Naturais e Matematica, Universidade Pedagogica, Campus de Lhanguene, Av. de Mogambique, 21402161 Maputo, Mogambique

(e) Centro de Malaria e Outras Doengas Trapicais (CMDT), Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal

* Corresponding author at: Faculty of Pharmacy, University of Lisbon, iMed.ULisboa, Natural Products Chemistry, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal. Tel.: +351 21 7946470; fax: +351 21 7946470.

E-mail address: mjuferreira@ff.ulisboa.pt (M. J. U. Ferreira).

URL: http://imed.ulisboa.pt/research/program-areas/drug-design/natural products-chemistry/ (M. J. U. Ferreira)

http://dx.doi.org/10.1016/j.phymed.2015.02.003

Table 1
Minimum inhibitory concentration (MIC), modulation factor (MF), and
fractional inhibitory concentration (FIC) values of antibiotics and
ethidium bromide alone and in combination with compounds 1-13, for S.
aureus ATCC 6538.

Compounds                                     Erythromycin
                                              (MIC = 0.2 [micro]g/ml)

                                              MIC     MF      FIC

Oxychelerythrine(l)                           0.1#    2#      0.5#
Oxynitidine (2)                               0.2     1       1
Arnottianamide (3)                            0.2     1       1
([+ or -])-Syringaresinol (4)                 0.2     1       1
(+)-Tembamide (5)                             0.2     1       1
(+)-Ailanthoidiol (6)                         0.2     1       1
(+)-Tembamide acetate (7)                     0.2     1       1
(+)-Ailanthoidiol diacetate (8)               0.2     1       1
(+)-Ailanthoidiol dibutanoate (9)             0.2     1       1
(+)-Ailanthoidiol di-2-methylbutanoate (10)   0.2     1       1
(+)-Ailanthoidiol di-2-ethylbutanoate (11)    0.2     1       1
(-F)-Ailanthoidiol didodecanoate (12)         0.2     1       1
(+)-Ailanthoidiol dibenzoate (13)             0.2     1       1

Compounds                                     Oxacillin
                                              (MIC = 0.2 [micro]g/ml)

                                              MIC     MF      FIC

Oxychelerythrine(l)                           0.05#   4#      0.25#
Oxynitidine (2)                               0.2     1       1
Arnottianamide (3)                            0.2     1       1
([+ or -])-Syringaresinol (4)                 0.2     1       1
(+)-Tembamide (5)                             0.2     1       1
(+)-Ailanthoidiol (6)                         0.2     i       1
(+)-Tembamide acetate (7)                     0.2     i       1
(+)-Ailanthoidiol diacetate (8)               0.2     i       1
(+)-Ailanthoidiol dibutanoate (9)             0.2     1       1
(+)-Ailanthoidiol di-2-methylbutanoate (10)   0.2     1       1
(+)-Ailanthoidiol di-2-ethylbutanoate (11)    0.2     1       1
(-F)-Ailanthoidiol didodecanoate (12)         0.2     1       1
(+)-Ailanthoidiol dibenzoate (13)             0.2     1       1

Compounds                                     Tetracycline
                                              (MIC = 0.1 [micro]g/ml)

                                              MIC     MF      FIC

Oxychelerythrine(l)                           0.05#   2#      0.5#
Oxynitidine (2)                               0.05#   2#      0.5#
Arnottianamide (3)                            0.05#   2#      0.5#
([+ or -])-Syringaresinol (4)                 0.1     1       1
(+)-Tembamide (5)                             0.1     1       1
(+)-Ailanthoidiol (6)                         0.1     1       1
(+)-Tembamide acetate (7)                     0.1     1       1
(+)-Ailanthoidiol diacetate (8)               0.1     1       1
(+)-Ailanthoidiol dibutanoate (9)             0.1     1       1
(+)-Ailanthoidiol di-2-methylbutanoate (10)   0.1     1       1
(+)-Ailanthoidiol di-2-ethylbutanoate (11)    0.1     1       1
(-F)-Ailanthoidiol didodecanoate (12)         0.1     1       1
(+)-Ailanthoidiol dibenzoate (13)             0.1     1       1

Compounds                                     Ethidium bromide
                                              (MIC = 4 [micro]g/ml)

                                              MIC     MF      FIC

Oxychelerythrine(l)                           2#      2#      0.5#
Oxynitidine (2)                               4       1       1
Arnottianamide (3)                            4       1       1
([+ or -])-Syringaresinol (4)                 4       1       1
(+)-Tembamide (5)                             4       1       1
(+)-Ailanthoidiol (6)                         4       1       1
(+)-Tembamide acetate (7)                     4       1       1
(+)-Ailanthoidiol diacetate (8)               1#      4#      0.25#
(+)-Ailanthoidiol dibutanoate (9)             4       1       1
(+)-Ailanthoidiol di-2-methylbutanoate (10)   2#      2#      0.5#
(+)-Ailanthoidiol di-2-ethylbutanoate (11)    2#      2#      0.5#
(-F)-Ailanthoidiol didodecanoate (12)         4       1       1
(+)-Ailanthoidiol dibenzoate (13)             4       1       1

The concentrations tested for compounds 1,4/13 were 100 [micro]g/ml,
and for compounds 2/3 were 50 [micro]g/ml. Values in bold correspond
to positive combination effect. Modulation factor (MF) =
[MIC.sub.(antibiotic)]/[MIC.sub.(antibiotic + modulator)]. Fractional
inhibitory concentration (FIC) = [MIC.sub.antibiotic incombination]/
[MIC.sub.antibiotic]

Values in bold correspond to positive combination effect indicated.
with #.

Table 2
Relative final fluorescence (RFF) values of compounds 1-13 and
verapamil for S. aureus ATCC 6538 as determined in real-time
fluorometry assays.

Compound                                 Concentration    Relative final
                                         ([micro]g/ml)    fluorescence
                                                          (RFF)

Verapamil                                200              0.55
(+)-Ailanthoidiol                        50               2.84
  di-2-ethylbutanoate (11)
(+)-Ailanthoidiol diacetate (8)          50               1.47
(+)-Ailanthoidiol dibutanoate (9)        50               1.00
(+)-Ailanthoidiol                        50               0.88
  di-2-methylbutanoate (10)
(+)-Ailanthoidiol(6)                     50               0.86
Oxynitidine (2)                          25               0.79
Oxychelerythrine(l)                      50               0.76
(+)-Tembamide acetate (7)                50               0.47
([+ or -])-Syringaresinol (4)            50               0.43
(+)-Ailanthoidiol didodecanoate (12)     50               0.39
(+)-Tembamide (5)                        50               0.31
(+)-Ailanthoidiol dibenzoate (13)        50               0.27
Arnottianamide (3)                       25               0.26

Values of relative final fluorescence (RFF) were calculated as
follows: RFF = [F.sub.compound] - [F.sub.control]/[F.sub.control],
using the fluorescence values taken at 60 min of assay. Values in bold
correspond to RFF values higher than the one for verapamil (0.55).

Values in bold correspond to RFF values higher than the one for
verapamil (0.55) indicated with #.

Table 3
Minimum inhibitory concentration (MIC), modulation factor (MF), and
fractional inhibitory concentration (FIC) values of ciprofloxacin and
EtBr alone and in combination with compounds 8 and 11.

Agent                             Ciprofloxacin

                                  Alone

Strain                            MIC([micro]g/ml)

S. aureus ATCC6538                0.25
S. aureus ATCC 25923              0.5
S. aureus ATCC [25923.sub.EtBr]   2
SMI                               256
SM39                              1

                                  Combination (8)

Strain                            MIC([micro]g/ml)   MF    FIC

S. aureus ATCC6538                0.25               1     1
S. aureus ATCC 25923              0.25#              2#    0.5#
S. aureus ATCC [25923.sub.EtBr]   1#                 2#    0.5#
SMI                               256                1     1
SM39                              0.25#              4#    0.25#

                                  Combination (11)

Strain                            MIC([micro]g/ml)   MF    FIC

S. aureus ATCC6538                0.125#             2#    0.5#
S. aureus ATCC 25923              0.25#              2#    0.5#
S. aureus ATCC [25923.sub.EtBr]   1#                 2#    0.5#
SMI                               256                1     1
SM39                              0.25#              4#    0.25#

                                  Alone

Strain                            MIC ([micro]g/ml)

S. aureus ATCC6538                4
S. aureus ATCC 25923              6.25
S. aureus ATCC [25923.sub.EtBr]   200
SMI                               16
SM39                              200

                                  Combination (8)

Strain                            MIC ([micro]g/ml)   MF    FIC

S. aureus ATCC6538                1#                  4#    0.25#
S. aureus ATCC 25923              1.56#               4#    0.25#
S. aureus ATCC [25923.sub.EtBr]   25#                 8#    0.125#
SMI                               4#                  4#    0.25#
SM39                              25#                 8#    0.125#

                                  Combination (11)

Strain                            MIC([micro]g/ml)   MF    FIC

S. aureus ATCC6538                2#                 2#    0.5#
S. aureus ATCC 25923              3.12#              2#    0.5#
S. aureus ATCC [25923.sub.EtBr]   100#               2#    0.5#
SMI                               16                 1     1
SM39                              200                1     1

Modulation factor (MF) = MIC (antibiotic)/MIC (antibiotic +
modulator). Fractional inhibitory concentration (FIC) =
[MIC.sub.antibiotic in combination]/[MIC.sub.antibiotic]. Compounds 8
and 11 were tested at concentration of 100 [micro]g/ml. Values in bold
correspond to positive combination effect.

Values in bold correspond to positive combination effect indicated
with #.

Table 4
Relative final fluorescence (RFF) values of compounds 8 and 11 and
verapamil for S. aureus strains ATCC 25923, ATCC [25923.sub.EtBr], SMI
and SM39, as determined in real-time fluorometry assays.

Compound                      Concentration
                              ([micro]g/ml)

Verapamil                     200
(-p)-Ailanthoidiol            50
  di-2-ethylbutanoate (11)
(+)-Ailanthoidiol diacetate   50
  (8)

Compound                      Relative final fluorescence (RFF)

                              ATCC    ATCC               SMI    SM39
                              25923   [25923.sub.EtBr]

Verapamil                     0.76    0.64               1.95   0.86
(-p)-Ailanthoidiol            0.98#   0.10               0.33   0.20
  di-2-ethylbutanoate (11)
(+)-Ailanthoidiol diacetate   0.84#   0.00               0.38   0.30
  (8)

Values of relative final fluorescence (RFF) were calculated as
follows: RFF = [F.sub.compound] - [F.sub.control]/[F.sub.control,
using the fluorescence values taken at 60 min of assay. Values in bold
correspond to RFF values higher than the one for verapamil (0.55) in
strain S. aureus ATCC 6538.

Values in bold correspond to RFF values higher than the one for
verapamil (0.55) in strain S. aureus ATCC 6538 indicated with #.
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Author:Cabral, Vanessa; Luo, Xuan; Junqueira, Elisabete; Costa, Sofia S.; Mulhovo, Silva; Duarte, Aida; Cou
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUPR
Date:Apr 15, 2015
Words:8645
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