Microbial Pathogenesis 104 (2017) 151e154

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Modulation of the antibiotic activity against multidrug resistant

strains of coumarins isolated from Rutaceae species
Sara A.L. Madeiro a, Nathalie H.P.B. Borges b, Augusto L. Souto c, Pedro T.R. de Figueiredo c,

P. Siqueira-Junior b, Josean F. Tavares c, *
Jose
a ~o Pessoa, Brazil
Centro de Biotecnologia, Universidade Federal da Paraíba, Joa
b
Departamento de Biologia Molecular, Universidade Federal da Paraíba, Joa ~o Pessoa, Brazil
c
Departamento de Ci^encias Farmac^ ~o Pessoa, Brazil
euticas, Universidade Federal da Paraíba, Joa

a r t i c l e i n f o a b s t r a c t

Article history: The first occurrences and dissemination of resistant microorganisms led to the inefficacy of many an-
Received 6 December 2016 tibiotics, available in the market nowadays, therefore, the search for new substances with antimicrobial
Received in revised form activity from natural sources has gained a great importance. The purpose of this work is to evaluate the
10 January 2017
antibacterial activity and modulation of drug resistance in Staphylococcus aureus by coumarins such as
Accepted 18 January 2017
Available online 19 January 2017
bergapten, xantotoxin, isopimpinellin and imperatorin obtained from two Rutaceae species (Metrodorea
mollis and Pilocarpus spicatus). The antimicrobial activity was assessed based on the minimum inhibitory
concentration (MIC), using the microdilution method. The MIC was >256 g/mL for all coumarins tested.
Keywords:
Modulation
Regarding the modulation of drug resistance assay, the isopimpinellin reducted the MIC of erytromicin
Microbial resistance by 4 times, whereas imperatorin exhibited the best result, reducing the MIC of tetracycline (2 times),
Coumarins erytomicin (4 times) and norfloxacin (4 times). By reducing the MIC of ethidium bromide, the imper-
Rutaceae atorin is consider in fact, as a putative efflux pump inhibitor of bacteria.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction attractive strategy in order to reduce the resistance of bacteria to

The arising and dissemination of resistant microorganisms
made some antibiotics, available at the market nowadays, become
ineffective [1]. Due the issues related to multidrug bacterial resis-
tance, the search for new drugs with antimicrobial activity from
natural sources has gained great importance [2,3]. The interest for
plants, come partially from a wide variety of chemical compounds,
belonging to different classes of secondary metabolites, such as
terpenoids, coumarins, flavonoids and alkaloids, many of which,
have potent antimicrobial activity already reported by previous
research [4,5]. The compounds isolated from plants have different
chemical structures when compared to antimicrobial compounds
obtained from bacteria, yeasts and fungi. Besides its direct anti-
microbial activity, these natural compounds have been studied as
modulator agents of antibiotic activity, increasing the activity of
antibiotics against bacterial strains resistant to these drugs. The
development of these modulator agents represents a quite
* Corresponding author.
E-mail address: josean@ltf.ufpb.br (J.F. Tavares).
drugs, once it could facilitate antibiotic recycling, which are
frequently cheaper and less toxic than new antimicrobial agents
[6,7]. Therefore, in order to search for bioactive natural products,
this work will describe the antibacterial activity and modulation of
drug resistance in Staphylococcus aureus by the coumarins: ber-
gapten, xantotoxin, isopimpipinellin and imperatorin (obtained
from Pilocarpus spicatus and Metrodorea mollis species) determined
by the minimum inhibitory concentration (MIC).
2. Materials and methods
2.1. Coumarins
Coumarins were isolated from the aerial parts of M. mollis and
P. spicatus species, which were harvested in june of 2011, at the city
of Mature
ia, located at the state of Paraíba, Brazil. The botanical
material was identified by Prof. Dr. Maria de Fa
tima Agra, which
deposited a voucher specimen of each species at the herbarium
Prof. Lauro Pires Xavier (JPB) encoded as 7449 and 7428 respec-
tively. The aerial parts of each species were oven-dried at 45
C,
ground and macerated with hexane, dichloromethane, ethyl

http://dx.doi.org/10.1016/j.micpath.2017.01.028
0882-4010/© 2017 Elsevier Ltd. All rights reserved.
152 S.A.L. Madeiro et al. / Microbial Pathogenesis 104 (2017) 151e154

OCH3

O
O O O
O O

OCH3

Bergapten Xantotoxin
OCH3

O O
O O O O

OCH3 O

Isopimpinellin Imperatorin

Fig. 1. Coumarins isolated from M. mollis and P. spicatus.

acetate and methanol in order to obtain its corresponding extracts.
The hexanic extract of M. mollis was submitted to Medium Pressure
Liquid Chromatography (MPLC), using column with silica gel 60
(0.063e0.2 mm) as stationary phase and the solvents hexane,
dichloromethane (CH2Cl2) and ethanol (MeOH) as mobile phase
with a 30 mL/min flow. The fractions were analyzed by thin layer
chromatography (TLC) and reunited according to their respective
retention factors (RFs). The fraction 47e63 and 79e87 were indi-
cated as pure by TLC analysis and identified by NMR as the cou-
marins: xantotoxin and isopimpinellin respectively (Fig. 1). The
hexanic extract of P. spicatus was also submitted to MPLC, using the
same methodology described previously for M. mollis. The fraction
14e22 was submitted to Column Chromatography (CC), using silica
flash (0.02e0.063) as stationary phase and the eluents hexane,
AcOEt and MeOH as mobile phase. The coumarin bergapten was
obtained from the subfraction 10e12 of this column (Fig. 1). The
subfraction 6e9 was submitted to a new CC, using the same CC
methodology mentioned above which resulted in the subfraction
5e8, identified as imperatorin (Fig. 1).
has the efflux protein for tetracycline (TetK) [11]. All these strains
were maintained at blood agar base culture medium (BAB, Difco),
inclined. Before its use, the cells were inoculated and grown at
brain and heart infusion (BHI, Difco) for 18e24 h at 37
C.
2.3. Antibiotics and ethidium bromide
The antibiotics tetracyline, erytromicin and norfloxacin (Sigma-
Aldrich) were prepared according to de NCCLS e National Com-
mittee for Clinical Laboratory Standards [12]. Ethidium bromide, an
intercalating agent and nucleic acid stain, is also known as a sub-
strate for NorA efflux pump [13], ergo, it has a great importance for
the evaluation of modulation of drug resistance inSA-1199B strain.
The stock-solution of ethidium bromide was prepared with steril-
ized and distillated water.
Table 1
Minimum Inhibitory Concentration (MIC) of coumarins and antibiotics at mg/mL in
S. aureus strains that overexpress efflux pump.

Substances Strain (Efflux pump)

2.2. Microorganisms
IS-58 (TetK) RN4220 (MsrA) SA-1199B (NorA)

To evaluate the antibacterial activity and modulation of multi- Bergapten >256 >256 >256
drug resistance, the followed strains were provided by Prof. Simon Xantotoxin >256 >256 >256
Isopimpinellin >256 >256 >256
Gibbons (University of London) to conduct this experiment: SA-
Imperatorin >256 >256 >256
1199B, which overexpress the norA gene that encodes the NorA Tetracycline 64 e e
efflux protein, responsible for the efflux of non-structurally related Erytromicin e 512 e
drugs, including a class of fluorquinolones [8,9]; RN-4220 that has Norfloxacin e e 64
the pUL5054 plasmid, which carries the gene that encode the MsrA Chlorpromazinea 64 e 64
a
protein, correlated to the efflux of macrolides [10], and IS-58, which Positive control for TetK and NorA pump.
 S.A.L. Madeiro et al. / Microbial Pathogenesis 104 (2017) 151e154
Table 2
Minimum Inhibitory Concentration (MIC) of the antibiotics tetracycline, erythromycin and norfloxacin with and without subinhibitory concentrations of coumarins and
chlorpromazine (positive control).
Substances (at ¼ MIC) Strain (Efflux pump)/antibiotic (MICatmg/mL)
IS-58 (TetK)/Tetracycline (64)
Bergapten 64
Xantotoxin 64
Isopimpinellin 64
Imperatorin 32 (2)a
Chlorpromazin 32 (2)
a
(Fold reduction in MIC).
2.4. Drug susceptibility testing and modulation assay
The minimum inhibitory concentration (MIC) of antibiotics and
coumarins were determined by the microdilution assay in BHI us-
ing a bacterial suspension of 105 cfu/mL in saline solution of NaCl at
0,9%, with concentrations from 0,5e256 mg/mL (two fold serial
dilutions). The MIC was stablished as the lowest concentration that
completely inhibited bacterial growth after 24 h of cultivation at
37
C. The detection was done after the addition of resazurin at
0.01%. To evaluate the resistance modulation of coumarins, it was
determined the MIC of antibiotics and ethidium bromide with and
without subinhibitory concentrations of coumarins. All the assays
were conducted in duplicate and the results were expressed as a
mean of the replicates [4,14].
3. Results and discussion
The coumarins bergapten, xantotoxin, isopimpinellin and
imperatorin did not exhibit relevant antibacterial activity, pre-
senting a MIC >256 mg/mL (Table 1).
The utilization of mono and multicombinations of antibiotics
and natural products may affect not only one target but several,
where the different therapeutic components act in a synergic way
[15]. According to the data described at Table 2, it was observed that
the coumarins bergapten and xantotoxin did not modulate the
antibacterial activity of tetracycline, erythromycin and norfloxacin,
not demonstrating alteration of the MIC from the antibiotic tested
against the resistant strains evaluated. The isopimpinellin reduced
the MIC of erytromicin by 4-fold and did not exhibit any modifi-
cation effect of the antibacterial activity of the other two antibi-
otics. Among all the coumarins evaluated, the imperatorin
presented the best result regarding the modulation activity, with a
MIC reduction of tetracycline (two-fold), erytromicin (4-fold) and
norfloxacin (4-fold).
Comparing the chemical structures of the four coumarins eval-
uated, it was observed that the bergapten and xantotoxin have very
similar structures, with a slight difference at the position of the
methoxy group. The isopimpinellin presents two methoxy groups
whereas imperatorin have a prenyl group at the C-8. These sub-
stituents increase the lipophilic character of the molecule, facili-
tating its path through the bacterial membrane. This may be one of
the reasons why the last two compounds mentioned have
Table 3
Minimum Inhibitory Concentration (MIC) of EtBr with and without the coumarins
imperatorin towards the S. aureus AS-1199B (NorA) strain.
Substance MIC (mg/mL)
SA-1199B (NorA)
EtBr 32
EtBr þ Imperatorin (128 mg/mL) 4 (8)a
a
(Fold reduction in MIC).
153
RN4220 (MsrA)/Erythromycin (512) SA-1199B (NorA)/Norfloxacin (64)
>512 64
>512 64
128 (4) 64
128 (4) 16 (4)
>256 16 (4)
demonstrated better modulation activity, especially the imper-
atorin, as it is the most lipophilic one, as the lipophilicity of several
putative efflux pump inhibitors have been related at the literature
[5]. The presence of a prenyl group bonded to a carbocylic ring
enable the antimicrobial activity [16,17] and according to previous
studies, imperatorin have demonstrated antimicrobial activity of
wide spectrum towards several strains [18e21]. Considering that
the only coumarin that exhibited a modulation effect at the
inhibitory activity of the norfloxacin against S. aureus SA-1199B was
the imperatorin, this compound was also tested for the determi-
nation of the MIC in combination with ethidium bromide (EtBr).
The results of this assay, demonstrated an 8-fold reduction in the
MIC of EtBr (32 mg/mL to 4 mg/mL) (see Table 3). The EtBr is a well-
known substrate for the NorA efflux pump, once the only form of
acquired bacterial resistance by it, occurs by the efflux pump [22].
Therefore, the decrease of the MIC from EtBr indicates that the
resistance modulation to norfloxacin in S. aureus SA-1199B may
have occurred through the inhibition of the NorA efflux pump
system [14].
4. Conclusion
The coumarins (bergapten, xantotoxin, isopimpinellin and
imperatorin) did not exhibit any antibacterial activity, even in its
highest concentration used against S. aureus strains resistant to
tetracycline, erytromicin and norfloxacin by efflux pump mecha-
nism. Regarding modulation of antibiotic activity, the coumarins
bergapten and xantotoxin did not modulate antibacterial activity
against the strains evaluated, however, isopimpinellin reducted the
MIC of erythromycin and imperatorin presented the best modula-
tion activity, reducing the MIC of the three antibiotics evaluated,
furthermore, it was the only compound which modulated the
resistance to ethidium bromide. More detailed research are
necessary in order to enable the utilization of this natural product
combined to antimicrobial agents evaluated towards the patho-
genic strains. The results obtained at this work are quite promising,
which may stimulate future studies about the use of natural
products concerning the viability of its use against microbial
resistance.
Conflict of interest
The authors declare to have no conflict of interest regarding the
contents of this article.
Acknowledgement
We are very grateful to Prof. Simon Gibbons (University of
London) for his valuable and kind cooperation. This work was
supported by the Brazilian research agencies CNPq (470750/2013-
8) and CAPES (1763/2015).
154 S.A.L. Madeiro et al. / Microbial Pathogenesis 104 (2017) 151e154

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