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Microbiology
Chemotherapy 2011;57:305309
DOI: 10.1159/000328975
Phytochemical Prospection and
Modulation of Aminoglycoside Antibiotic
Activity by Croton campestris A.
E.F.F.Matias
a
K.K.A.Santos
a
T.S.Almeida
b
J.G.M.Costa
b
H.D.M.Coutinho
a


a
Laboratory of Microbiology and Molecular Biology, and
b
Laboratory of Natural Products Research,
University of the Region of Cariri, Crato , Brazil

Introduction
Escherichia coli is one of the principal causes of infec-
tious diseases in humans. These bacteria are known to
produce enterotoxins whose properties and role in diar-
rheal disease have been widely investigated. E. coli is not
only a very common intestinal pathogen but very often is
involved in extra-intestinal diseases in the intensive care
unit and in surgical wound infections. The activity of cy-
totoxins and their role in human infection has been iden-
tified [1] , mainly in infections of the urinary tract [2] .
Bacteria of the genus Staphylococcus are distributed in
nature, as well as being part of the normal microbiota of
the skin and of the mucosa of animals including birds.
Some specimens of Staphylococcus are frequently recog-
nized as etiologic agents of opportunistic infections in
many animals and humans [3] . S. aureus, S. epidermidis,
S. saprophyticus and S. haemolyticus are the most impor-
tant causative species of human and hospital infections.
Besides causing different types of intoxications, S. aureus
represents the most common etiologic agent of purulent
infections (e.g. furuncle, carbuncle, abscess, myocardi-
tis, endocarditis, pneumonia, meningitis, bacterial ar-
thritis) [4] .
In relation to pathogenic bacteria, the increase in bac-
terial resistance to antibiotics is a growing and worrisome
problem [5] . For patients, antimicrobial resistance in-
creases morbidity and mortality, while there is a signifi-
Key Words
Croton campestris A. Methanol extract Hexane extract
Antibacterial activity Modification of resistance
Antibiotics Staphylococcus aureus Escherichia coli
Abstract
Background: Escherichia coli is known to produce enterotox-
ins whose properties and role in diarrheal disease have been
extensively investigated; besides, this bacterium is related to
several extra-intestinal problems in the intensive care unit
and in surgical wound infections. Some species of Staphylo-
coccus are recognized as etiological agents of opportunistic
infections in animals and humans. This study is the first test
on the modulation of antibiotic activity by Croton campestris
A. against multiresistant strains of E. coli and Staphylococcus
aureus . Methods: In this study, the hexane and methanol ex-
tract of C. campestris A. was tested for antibacterial activi-
ty alone and in combination with aminoglycosides against
bacterial strains. The synergy of the methanol and hexane
extract was verified by microdilution method. Results: A
synergistic effect of both extracts combined with the amino-
glycosides was demonstrated. Conclusions: It is suggested
that the extracts from C. campestris A. could be used as a
source of natural product derived from this plant with re-
sistance-modifying antibacterial activity, providing a new
weapon against the problem of bacterial resistance to anti-
biotics. Copyright 2011 S. Karger AG, Basel
Received: August 6, 2010
Accepted after revision: January 13, 2011
Published online: July 20, 2011
H.D.M. Coutinho
Departamento de Cincias Biolgicas
Universidade Regional do Cariri
Rua Cel. Antonio Luis 1161, Pimenta, Crato, CE 63105-000 (Brazil)
Tel. +55 88 3102 1212, E-Mail hdmcoutinho @ gmail.com
2011 S. Karger AG, Basel
00093157/11/05740305$38.00/0
Accessible online at:
www.karger.com/che
Matias /Santos /Almeida /Costa /Coutinho

Chemotherapy 2011;57:305309 306
cant increase in costs for health care institutions [6] . With
respect to the growing clinical importance given to the
hospital community and bacterial infections and the pro-
gressive development of antimicrobial resistance, consid-
erable scientific research has focused on the antibacterial
properties of plant products [79] .
In the last years, there has been great scientific interest
in chemical and pharmacological investigations of the
biological properties of medicinal plants [1012] . Medic-
inal plants have been the source of many medications that
are now applied in clinical practice [13] .
The family Euphorbiaceae is made up of 317 genera
and about 7,500 species. The genus Croton, which has 700
species, is widely distributed in warm regions of the world
[14] . The species Croton campestris A., popularly known
as velame do campo, is a shrub originally from Brazil, oc-
curring mainly in the southeast and northeast regions
[15] , and it is widely used in traditional medicine as a
powerful depurative against scrophulosis, venereal dis-
eases, tumors, skin diseases, rheumatism, ulcers of the
uterus, diarrhea, arthritis and as a photosensitizing agent
[16, 17] .
With the increase in the incidence of resistance to an-
tibiotics, alternative natural products of plants could be
of interest [18] . Some plant extracts and phytochemicals
are known to have antimicrobial properties, which could
be of great importance for therapeutic treatments. In the
last years, various studies have been conducted in dif-
ferent countries, demonstrating the efficacy of this type
of treatment [19, 20] . Many plants have been evaluated
not only for direct antimicrobial activity but also as re-
sistance-modifying agents [21] . Various chemical com-
pounds, synthetic or from natural sources, have direct
activity against many species of bacteria, enhancing the
activity of a specific antibiotic, reversing the natural re-
sistance of bacteria to specific antibiotics, causing the
elimination of plasmids and inhibiting the active efflux
of antibiotics through the plasma membrane [22] . The
potentiation of antibiotic activity or the reversal of anti-
biotic resistance allows the classification of these com-
pounds as modifiers of antibiotic activity [3, 22, 23] .
The aim of this study was to do a phytochemical
screening of the methanol and hexane extracts of C.
campestris A. and to determine their potentiation of the
antibiotic activity of aminoglycosides, an activity never
reported for this species.
Materials and Methods
Bacterial Material
The bacterial strains utilized were the clinical isolates E. coli
27 (EC27) and S. aureus 358 (SA358) with the resistance profile
described in table1 . All strains were maintained on slants with
heart infusion agar (Difco Laboratories Ltd.). Before the assay, the
cells were grown overnight at 37 C in brain heart infusion broth
(BHI; Difco Laboratories Ltd.).
Plant Material
Leaves of C. campestris A. were collected in the municipality
of Crato, Cear, Brazil. The plant material was identified and
dried, and pressed specimens were deposited in the Herbario da
Universidade Federal do Rio Grande do Norte, as No. 7095.
Preparation of Methanol and Hexane Extracts of
C. Campestris A.
For the preparation of extracts, leaves were collected which
were kept submersed in 800 ml of methanol and hexan separately
for 72 h; afterwards, the extract was filtered and concentrated us-
ing a rotary vacuum evaporator (model Q-344B, Quimis, Brazil)
and ultrathermal bath (model Q-214M2, Quimis), obtaining
31.2 g of leaf yields of 1.74 g of methanol extract and 71.38 g of
leaf yields of 0.87 g of hexane extract presented in table2 . The ex-
tract solutions utilized in the tests were prepared at a concentra-
tion of 10 mg/ml using dimethylsulfoxide (DMSO) and then di-
luted with distilled water to obtain a concentration of 1,024 g/ml.
Table 1. Bacterial source and antibiotic resistance profile
Bacteria Source Antibiotic resistance
EC27 surgical
wound
Ast, Ax, Amp, Ami, Amox, Ca, Cfc, Cf,
Caz, Cip, Chlo, Im, Kan, Szt, Tet, Tob
SA358 surgical
wound
Oxa, Gen, Tob, Ami, Kan, Neo, Para,
But, Sis, Net
A st = Aztreonam; Ax = amoxacilin; Amp = ampicillin; Ami =
amikacin; Amox = amoxicillin; Ca = cefadroxil; Cfc = cefaclor;
Cf = cefalotin; Caz = ceftazidime; Cip = ciprofloxacin; Chlo =
chloramphenicol; Im = imipenem; Kan = kanamycin; Szt = sul-
fametin; Tet = tetracycline; Tob = tobramycin; Oxa = oxacillin;
Gen = gentamicin; Neo = neomycin; Para = paromomycin; But =
butirosin; Sis = sisomicin; Net = netilmicin.
Table 2. Dry mass and yield of methanol and hexane extracts
Species Solvent used Leaves Yield
Croton campestris A. MECC 31.2 1.74
HECC 72.38 0.87
D ata are given in grams.
Modulation of Antibiotic Activity Chemotherapy 2011;57:305309 307
Phytochemical Prospecting
Phytochemical tests to detect the presence of heterosides, sa-
ponins, tannins, flavonoids, steroids, triterpenes, cumarins, qui-
nones, organic acids and alkaloids were performed according to
the method described by Matos [24] . The tests were based on the
visual observation of a change in color or formation of precipitate
after the addition of specific reagents, and the results for the ex-
tracts studied are shown in table3 .
Drugs
Gentamicin, kanamycin and amikacin were obtained from
Sigma Chemical Co. All drugs were dissolved in sterile water.
Modulation of Antibiotic Activity
The minimal inhibitory concentration (MIC) was determined
in a microdilution assay [25] utilizing an inoculum of 100 l of
each strain, suspended in BHI broth up to a final concentration
of 10
5
CFU/ml in 96-well microtiter plates, using 2-fold serial di-
lutions. Each well received 100 l of each extract solution. The
final concentrations of the extracts varied between 8 and 512 g/
ml. MICs were recorded as the lowest concentrations required to
inhibit growth. The MIC for the antibiotics was determined in
BHI by the microdilution assay utilizing suspensions of 10
5
CFU/
ml and a drug concentration range of 0.00122.5 mg/ml (2-fold
serial dilutions) [25] . The MIC was defined as the lowest concen-
tration at which no growth was observed. For the evaluation of
the extracts as modulators of resistance to the antibiotics, MIC
of the antibiotics was determined in the presence or absence of
methanol extract of C. campestris (MECC) and hexane extract of
C. campestris (HECC) at subinhibitory concentrations (8 g/ml),
and the plates were incubated for 24 h at 37 C. Each antibacterial
assay for MIC determination was carried out in triplicate, and
controls using DMSO for MIC and antibiotic modifying activity
tests were performed.
Results
Table3 shows that phytochemical prospection detect-
ed the presence of compounds such as phenols, tannins
pyrogallates and phlobaphenes, anthocyanins, anthocy-
anidins, flavones, flavonols, xanthones, chalcones, au-
rones, flavononols, leucoanthocyanidins, catechins, fla-
vonones, alkaloids and terpenes.
Table4 shows the MICs of the extracts and the poten-
tiating effect in combination with antibiotics. The strain
Table 3. Phytochemical prospection of MECC and HECC
Extracts M etabolites
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
MECC + + + + + + + + + +
HECC + + + + + + + + + +
1 = Phenols; 2 = tannin pyrogallates; 3 = tannin phlobaphenes; 4 = anthocyanins; 5 = anthocyanidins; 6 =
flavones; 7 = flavonols; 8 = xanthones; 9 = chalcones; 10 = aurones; 11 = flavononols; 12 = leucoanthocyanidins;
13 = catechins; 14 = flavonones; 15 = alkaloids; 16 = terpenes; + = presence; = absence.
Table 4. MIC values (g/ml) of aminoglycosides in the absence and presence of 8 g/ml of MECC and HECC
against EC27 and SA358
Antibiotics EC27 SA358
MIC alone MIC combined MIC alone M IC combined
MECC HECC MECC HECC
Gentamicin 19 (R) 9 (R) 2.2 (S) 39 (R) 2.2 (S) 2.2 (S)
Kanamycin 157 (R) 157 (R) 19 (R) 317 (R) 78 (R) 4.5 (S)
Amikacin 157 (R) 9 (S) 39 (R) 78 (R) 9 (S) 4.5 (S)
MECC 512 1,024
HECC 256 1,024
R = Phenotypic profile of resistance according to the CLSI; S = phenotypic profile of sensitivity according to
the CLSI.
Matias /Santos /Almeida /Costa /Coutinho

Chemotherapy 2011;57:305309 308
EC27 showed greater sensitivity to the extracts than
strain SA358, but when antibiotics were combined with
the extracts, the strain SA358 showed greater sensitivity.
We found that HECC and the combination HECC-anti-
biotic was more effective. This finding can be due to the
presence of compounds with known antibacterial activ-
ity and with nonpolar characteristics, such as tannins,
flavonols and terpenes, extracted mainly by nonpolar sol-
vents such as hexane. Against the Gram-negative strain,
HECC showed synergism when combined with all the
antibiotics tested, while MECC showed no effect on
kanamycin activity ( table4 ). Against strain SA358, syn-
ergism was observed with all antibiotics for both extracts
tested ( table4 ). The control using DMSO showed a MIC
6 1,024 g/ml and no modifying antibiotic activity.
Discussion
Through phytochemical prospecting of the extracts, it
was possible to determine the presence of diverse classes
of secondary metabolites that show a wide variety of bio-
logical activities such as antimicrobial [26] , antioxidant
[27] , antitumor and antiophidic [28] and photosensitiz-
ing [17] .
In the tannins, the antimicrobial properties appear to
be associated with the hydrolysis of an ester bond with
gallic acid, thereby serving as a mechanism of natural de-
fense against microbial infections. The antimicrobial
property of tannic acid can also be utilized in food pro-
cessing to increase shelf life. The tannin components of
epicatechin and catechin ( Vaccinium vitisidaea L.) dem-
onstrated strong antimicrobial activity against bacteria
and fungi [29] .
Flavonoids are synthesized by plants in response to
microbial infection [30] and are effective against a broad
range of microorganisms. The activity is probably due to
their capacity to form complexes with extracellular solu-
ble proteins, which bind to the bacterial cell wall. Some
lipophilic flavonoids can also cause rupture of the plasma
membrane of microorganisms [31] .
Terpenes occur in the form of diterpenes, triterpenes,
tetraterpenes as well as hemiterpenes and sesquiterpenes.
Terpenenes or terpenoids are active against bacteria [32] .
The seeds contain active components; for example, vol-
atile oil and thymoquinone afford protection against
nephrotoxicity and hepatotoxicity induced by any disease
or chemical product [33] .
The mechanisms by which extracts can inhibit the
growth of microorganisms are varied and can be due in
part to the hydrophobic nature of some components. As
a result, they can show greater interaction with the lipid
bilayer of the cell membrane, affecting the respiratory
chain and the production of energy [34] , or even make the
cell more permeable to antibiotics, leading to the inter-
ruption of vital cellular activity [35] . Various components
of extracts can permeabilize the cell membrane, increas-
ing the penetration of antibiotics [36] . The interference
with bacterial enzyme systems can also be a potential
mechanism of action [37] . These mechanisms of action
can be obtained by the combination of antibiotic with ex-
tract at a subinhibitory concentration applied directly to
the culture medium [11, 12] . This is indicated by the phe-
notypic reversion observed using the breakpoints indi-
cated by the CLSI [25] . The HECC was more effective in
changing the resistant phenotype of the bacterium to the
sensitive one. This capacity was more evidenced in the
Gram-positive bacteria, indicating that this fact could be
related with the polarity of extract and with the bacterial
type. Several plants showed the capacity of modifying the
antibiotic activity, as Mentha arvensis, Turnera ulmifolia,
Momordica charantia and others [3, 11, 22] , but this is the
first report of this activity in a plant of the genus Cordia
and in the C. campestris species.
This strategy is called herbal shotgun or synergistic
multi-effect targeting and refers to the utilization of
plants and drugs in an approach using mono- or multi-
extract combinations, which can affect not only a single
target but various targets, where the different therapeutic
components collaborate in a synergistic-agonistic man-
ner. This approach is not only for combinations of ex-
tracts; combinations between natural products or ex-
tracts and synthetic products or antibiotics are also pos-
sible [3840] .
The results obtained indicate that C. campestris A.
could serve as a source of plant-derived natural products
that modify antibiotic resistance for use against multi-
drug-resistant bacteria, such as strains of E. coli and
S. aureus acquired from the hospital and from the com-
munity.
Acknowledgements
The authors are grateful to the Brazilian research agencies
FUNCAP and CNPQ.

Modulation of Antibiotic Activity Chemotherapy 2011;57:305309 309
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