1265

Introduction
Te incidence of community and hospital infections
associated with Staphylococcus aureus has increased.
S. aureus is a pathogen with a high virulence, given its
capacity of systemic dissemination, where it is frequently
related to infections from wounds, pneumonia, septice-
mia and endocarditis. Patients, especially those hospital-
ized in an intensive care environment, are susceptible to
bacteremia, particularly involving methicillin-resistant S.
aureus (Selvey et al., 2000; Gibbons, 2004).
Escherichia coli is a Gram-negative bacillus that causes
infections, especially neonatal, such as meningitis and sep-
ticemia, and even diarrheal diseases, in the whole world,
particularly afecting children up to 5 years old. It is found
in the vaginal and intestinal fora, thereby being implicated
in a variety of infectious diseases. It is believed that vaginal
contamination during birth is an important source of neo-
natal infection (Krohn et al., 1997; Bingen et al., 1998).
Microbial resistance to drugs employed in the routine
treatment of infections has increased morbidity and
RESEARCH ARTICLE
Antibacterial and modulatory efect of Stryphnodendron
rotundifolium
Dayanne R. Oliveira
1
, Francisco E. Brito-Junior
1
, Elizngela B. Bento
1
, Edinardo F.F. Matias
2
, Ana
Carla A. Sousa
2
, Jose G.M. Costa
3
, Henrique D.M. Coutinho
2
, Marta R. Kerntopf
1
, and Irwin R.A.
Menezes
1
1
Laboratory of Pharmacology and Molecular Chemistry, Regional University of Cariri, Crato, Brazil,
2
Laboratory of
Microbiology and Molecular Biology, Regional University of Cariri, Crato, Brazil, and
3
Laboratory of Research in Natural
Products, Regional University of Cariri, Crato, Brazil
Abstract
Context: Stryphnodendron rotundifolium Mart. (Leguminosae), a tree in Northeast Brazil (Chapada do Araripe), is used
in popular medicine to treat diferent processes such as infammation and infectious diseases, mainly caused by
bacterial pathogens.
Objective: This study determined the modulatory and antimicrobial activity of the hydroethanol extract of dried stem
bark, the most used form of this natural product, as a remedy by the traditional communities, against standard and
clinical isolates of Escherichia coli and Staphylococcus aureus.
Material and methods: The antibacterial and modulatory activities of the hydroalcoholic extract from the leaves were
obtained by maceration/hydrodistillation method and assayed by microdilution.
Results: In the microbiological assays, growth inhibition was demonstrated by this extract against the bacterial
strains tested, with minimal inhibitory concentration (MIC) values of 512 g/mL. However, when a subinhibitory
concentration (MIC/8 = 64 g/mL) was combined with conventional antimicrobial drugs (gentamicin, kanamycin,
amikacin and neomycin), the extract showed a potentiating efect, reducing the MIC for all drugs assayed in a range
between 312.5 and 2.4 g/mL.
Conclusions: We indicate that the extract of S. rotundifolium showed potential synergistic antibiotic activity. With the
results obtained, these extracts proved to be a promising source of antibacterial and modulatory agents.
Keywords: Aminoglycosides, antimicrobial activity, multidrug resistant, tannins; favonoids
Address for Correspondence: H.D.M. Coutinho, Laboratory of Microbiology and Molecular Biology LMBM, Department of Biological
Chemistry, Regional University of Cariri URCA, Cel. Antonio Luis Street, 1161, Pimenta 63105-000, Crato (CE), Brazil. Tel.:
+55 88 31021212. Fax: +55 88 31021291. E-mail: hdmcoutinho@gmail.com
(Received 24 January 2011; revised 21 April 2011; accepted 04 May 2011)
Pharmaceutical Biology, 2011; 49(12): 12651270
2011 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2011.589857
Pharmaceutical Biology
2011
49
12
1265
1270
24 January 2011
21 April 2011
04 May 2011
1388-0209
1744-5116
2011 Informa Healthcare USA, Inc.
10.3109/13880209.2011.589857
NPHB
589857
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1266 D.R. Oliveira et al.
Pharmaceutical Biology
mortality, raising hospital costs, which has demonstrated
the importance of the discovery of new drugs. Terefore,
natural products appear to be an interesting alternative.
Te use of medicinal plants with antimicrobial potential
or that can serve as modifers of antibiotic activity has
stirred the interest of investigators with the aim of discov-
ering new drugs for the treatment of multidrug resistant
infections (Lu et al., 2007; Ennajar et al., 2009; Coutinho
et al., 2010).
Te genus Stryphnodendron Mart. (Leguminosae)
is composed of about 48 species mentioned in the lit-
erature; all are native to the Brazilian Cerrado. Studies of
these species have demonstrated that stem bark consists
of 20% tannins, which is recommended in popular medi-
cine for various afictions (Cronquist, 1988; Santos &
Mello, 2004). Species of this genus are utilized in popular
medicine for leukorrhea and gynecologic problems, as an
antibacterial, antihemorrhagic, antihypertensive or anti-
ulcerogenic, for diarrhea and infammatory processes, as
an astringent, antiseptic or diuretic and for cleansing and
healing of wounds, especially in the form of a decoction
(Vasconcelos et al., 2004; Souza et al., 2007). Te biologi-
cal studies of the genus show that Stryphnodendron has
several activities: antimicrobial (Sanches et al., 2005),
antiulcer (Rodrigues et al., 2008), antiviral (Felipe et al.,
2006), trypanocidal (Holetz et al., 2005) and against
Candida albicans (Ishida et al., 2006).
Stryphnodendron rotundifolium, popularly known
as barbatimo, is a plant species that is endemic to the
Cerrado area of Chapada do Araripe, in Northeast Brazil
(Correia, 1984). It is utilized for a variety of health prob-
lems by local communities, particularly by the inhabit-
ants of rural areas. Te popular use of this plant, for
which there is a lack of scientifc investigation, justifes
the need for studies to explore the biological potential of
the plant.
Tis work carried out a phytochemical screening of
the hydroethanol extract of dried stem bark of S. rotun-
difolium to investigate the extract for antibacterial activ-
ity and antibiotic-modifying efects, and to correlate the
fndings with the popular use of this species.
Methods
Drugs
Gentamicin, amikacin, kanamycin and neomycin were
from Sigma Co. (St. Louis, MO). Te solutions of the anti-
biotics were prepared based on the recommendations of
the Clinical and Laboratory Standards Institute (NCCLS,
2008).
Bacterial strains
Te following bacterial strains were used: E. coli (EC27)
and S. aureus (SA358) with a drug resistance profle
described in Table 1. All strains were maintained on heart
infusion agar slants (Difco Laboratories Ltd). Before
assays, bacterial cells were grown overnight at 37C in
brain heart infusion broth (BHI; Difco Laboratories Ltd).
Plant material
Te stem bark of the plant species S. rotundifolium
was collected in March 2010 (rainy season) from the
municipality of Crato, Ceara, Brazil, using a GPS device.
Te following coordinates were obtained: 7 22 2.8 S;
39 28 42.4 W; the altitude was 892 m above sea level.
Te botanic material was identifed by Dr. Maria Arlene
Pessoa da Silva and a dried specimen of the species
deposited as #4661 in the Herbario Caririense Dardano
de AndradeLima of the Universidade Regional do
CaririURCA.
Preparation of hydroalcoholic extract of the bark of
S. rotundifolium
Te plant material (dried stem bark3710.5 g) was
extracted in the cold water in a thermal bath, using 99.9%
ethanol in a 1:1 proportion (8.7 L of solvent) for 72 h. Te
extracts were fltered and vacuum-concentrated using
a rotary evaporator, yielding a crude lyophilizate of the
extracts, as shown in Table 2. For the tests, the extracts
were diluted in dimethyl sulfoxide at a concentration of
10 mg/mL, followed by dilution in distilled water to give
a concentration of 1024 g/mL.
Phytochemical prospecting
Phytochemical prospecting for the detection of tannins,
favonoids, saponins, steroids, triterpenes, cumarins,
quinones, organic acids and alkaloids was carried out
according the method described by Matos (1997). Te
results are given in Table 3.
Antimicrobial activity test
Te minimal inhibitory concentration (MIC) of each
extract was determined in a microdilution assay (NCCLS,
2008), utilizing an inoculum of 100 L of each bacterial
strain, suspensions in BHI broth up to a fnal concentration
of 10
5
colony-forming units/mL in 96-well microdilution
plates with serial twofold dilutions. A volume of 100 L
of each extract solution was added to each well. Te fnal
concentrations of the extracts varied from 512 to 8 g/mL.
MIC was determined as the least concentration that inhib-
ited bacterial growth. Te potentiating efect of the extracts
Table 1. Bacterial source and antibiotic resistance profle.
Bacteria Source Antibiotic resistance
Escherichia coli 27 Surgical wound Ast, Ax, Amp, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Im, Can, Szt, Tet, Tob
Staphylococcus aureus 358 Surgical wound Oxa, Gen, Tob, Ami, Can, Neo, Para, But, Sis, Net
Ast, aztreonam; Ax, amoxacilin; Amp, ampicillin; Ami, amikacin; Amox, amoxicillin; Ca, cefadroxil; Cfc, cefaclor; Cf, cefalotin; Caz,
ceftazidime; Cip, ciprofoxacin; Chlo, chloranphenicol; Im, imipenem; Kan, kanamycin; Szt, sulfametim; Tet, tetracycline; Tob,
tobramycin; Oxa, oxacillin; Gen, gentamicin; Neo, neomycin; Para, paramomycin; But, butirosin; Sis, sisomicin; Net, netilmicin.
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Activity of Stryphnodendron rotundifolium 1267
2011 Informa Healthcare USA, Inc.
was tested against strains of E. coli (EC27) and S. aureus
(SA358) in combination with the following antimicrobial
agents: gentamicin, kanamycin, neomycin and amikacin
at fnal concentrations varying from 1024 to 1 g/mL after
serial two-fold dilutions as cited before. Te plates were
incubated under aerobic conditions for 24 h at 37C.
Results
A preliminary ethnopharmacologic study revealed that
S. rotundifolium is used as a popular medicine by tradi-
tional communities, where the stem bark of this species
represents the part of the plant recommended for use,
especially in the form of a decoction. Tis medicinal
plant is used in treating a variety of ailments, including
wounds and infections, gastric disturbances, infam-
mation, painful conditions, dermatoses, pulmonary
disease, diseases of the female reproductive tract and
diseases of the prostate (data not shown). Phytochemical
prospecting of hydroalcoholic extract of S. rotundifolium
(HESR) allowed the identifcation of tannins, pyrogallics,
favones, favonoids, favononols, xanthones, chalcons,
favonones and steroids (Table 3).
HESR demonstrated antimicrobial activity of little sig-
nifcance against the bacterial strains tested, with MIC
values of 512 g/mL. When HESR at a concentration
of 64 L/mL was combined with the aminoglycosides
(Table 4), it demonstrated a substantial potentiating
efect in relation to the antibacterial action of the drugs
alone, reducing the concentration necessary to inhibit
the bacterial growth.
Discussion
Te rise in the occurrence of multidrug resistant infec-
tions has stimulated research into the evaluation of
natural products for antimicrobial activity (Arango et al.,
2004). Various biological properties are attributed to
the genus Stryphnodendron sp., and in vitro assays with
extracts of species of this genus have shown antibacterial
activity (Audi et al., 2004; Holetz et al., 2005).
Phenolic compounds, including tannins and fa-
vonoids, have demonstrated their therapeutic poten-
tial as anti-infammatory, antifungal, antimicrobial,
antioxidant and wound-healing agents (Santos et al.,
2004). Data from the literature confrm the use of stem
bark of species of the genus Stryphnodendron for vari-
ous ailments. Investigations to determine the efect of
bark extracts of species of this genus have demon-
strated the presence of tannins and favonoids, their
use as antimicrobial agents (Matos, 1997; Singh et al.,
2006; Lopes et al., 2003). Fractions of the extracts were
also used for the identifcation of antibacterial proper-
ties against S. aureus and Gram-negative bacteria, also
demonstrating a growth-inhibitory efect and antiradi-
cal properties. Tis species contains 1219% tannins in
the bark of stems (Lopes et al., 2003). Te precise profle
of active constituents of S. rotundifolium is still not well
known; however, tannins can be considered the most
relevant substances due to its high content. Te chemi-
cal compositions of the Stryphnodendron genus indicate
the presence of sapogenines and proanthocyanidins
(Tursch et al., 1963), gallocatechin (Reicher et al., 1992)
and monomeric, dimeric or trimeric condensed tannins
(Lopes et al., 2009). Tree species of Stryphnodendron
genus [S. adstringens (Mart.) Coville, S. polyphyllum
and S. obovatum Benth.] demonstrated that the main
compounds in ethanol extract are gallic acid and gallo-
catechin. Te study performed by Mello et al. (1996a,b)
showed that the concentration of gallic acid is variable.
However, the concentration of gallocatechin is similar
between the three species.
Table 2. Dry mass and yield of hydroalcoholic extracts (g).
Species Solvent used Leaves Yield
Stryphnodendron
rotundifolium
Mart.
HESR 3710.5 627.8
HESR, hydroalcoholic extract of Stryphnodendron rotundifolium
Mart.
Table 3. Phytochemical prospection of hydroalcoholic extract of Stryphnodendron rotundifolium Mart.
Metabolites
Extracts 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
HESR + + + + + + + + +
1, phenols; 2, tannin pyrogallates; 3, tannin phlobaphenes; 4, anthocyanins; 5, anthocyanidins; 6, favones; 7, favonols; 8, xanthones;
9, chalcones; 10, aurones; 11, favononols; 12, leucoanthocyanidins; 13, catechins; 14, favonones; 15, alkaloids; 16, terpenes; 17,
steroids; (+), presence; (), absence.
Table 4. MIC values (g/mL) of aminoglycosides in the absence and presence of 64 g/mL of HESR against Escherichia coli 27 and
Staphylococcus aureus 358.
EC27 SA358
Drugs and extract alone Drugs + HESR Drugs and extract alone Drugs + HESR
Gentamicin 2500 19.5 39 2.4
Kanamycin 2500 62.5 2500 312.5
Amikacin 156.2 9.7 78.1 9.7
Neomycin 312.5 39 312.5 9.7
HESR 512 512
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1268 D.R. Oliveira et al.
Pharmaceutical Biology
Susceptibility tests using the difusion method
showed that Stryphnodendron adstringens was active
against Streptococcus mutans, S. aureus, Actinobacillus
actinomycetemcomitans and C. albicans, but that only
the ethanol extract also had antifungal activity (Santos
et al., 2002). However, it is important to mention that the
microdilution method, employed in the present investi-
gation, currently represents the technique most accepted
for this bioassay (Hadacek & Greger, 2000).
Other studies with fractions of extracts of the stem
bark of S. adstringens, using the microdilution tech-
nique, revealed antifungal activity against Candida
spp., attributed to level of tannins present in this species
(Ishida et al., 2006). In this context, the evaluation of the
antioxidant and antimicrobial activities of the bark of S.
adstringens, by the dilution assay, can verify the presence
of antioxidant and antimicrobial properties, where both
bioactive actions are associated with the presence of total
phenolic compounds, such as tannins and favonoids
(Souza et al., 2007).
Assays carried out with extracts of plants rich in tan-
nins have demonstrated bactericidal and fungicidal
efects (Scalbert, 1991), antiviral activity (De Bruyne et al.,
1999), inhibition of enzymes such as glycosyltransferases
of S. mutans and Streptococcus sobrinus (Ooshima et al.,
1993) and antioxidant activity (Moure et al., 2001). Te
capacity of tannins to form complexes with proteins is
accepted as the basis for protection against insects, fungi
and bacteria, as well as their pharmacological proper-
ties. Te exact mechanisms by which these compounds
exert their antimicrobial efects have not been well elu-
cidated. However, three hypotheses have been proposed.
One would be the inhibition of enzymes of bacteria and
fungi and/or complexation of their substrates; another
mechanism would be represented by their action on
the cell membranes of microorganisms, thereby altering
metabolism; and the third would be complexation with
metal ions (Mila et al., 1996), reducing the availability of
these elements in metabolism (Ho et al., 2001; Schofeld
et al., 2001; Santos & Mello, 2004).
Tere was a signifcant diference between gallocat-
echin and epigallocatechin, which may be related to the
stereochemistry of the compounds. Note the presence of
a methoxyl group at position 4 of gallocatechin, which
reduced its free-radical scavenging activity compared to
the non-substituted compound. Tere was a signifcant
diference between dimers with a hydroxyl group at posi-
tion 5 (prodelphinidins) and those without a hydroxyl
group at this position (prorobinetinidins). Tis confrms
the observation of van Acker et al. (1996) that compounds
with groups of the pyrogallol type (trihydroxylated) pos-
sess greater activity than compounds with groups of
the catechol type (dihydroxylated). However, this is the
frst report to compare antioxidant activity between
proanthocyanidins with or without a hydroxyl group at
a ring position 5. For this evaluation, there are no cur-
rently available parameters for comparison because
5-deoxyproanthocyanidins have been isolated from only
a few plant species, including Stryphnodendron spp. (van
Acker et al., 1996).
Salazar-Aranda et al. (2009) showed that extract
from Ceanothus coeruleus displayed the greatest activ-
ity against yeast as well as the best antioxidant activity.
Seven (46.6%) of the 15 extracts that showed antioxidant
activity displayed antibacterial activity as well, and nine
(80%) resulted active against yeast. Te study of Belofsky
et al. (2004) demonstrated an increase in the antimicro-
bial activity of pure compounds when they are combined
with antioxidants. Terefore, we consider that if both
antimicrobial and antioxidant compounds exist in the
extracts, they could interact and enhance the antimicro-
bial activity.
A variety of studies have demonstrated the antimi-
crobial and antifungal properties of favonoids (Rauha
et al., 2000; Sohn et al., 2004). Tese compounds have
the capacity to form complexes with extracellular soluble
proteins which bind to the bacterial cell wall (Tsuchiya
et al., 1996). Studies have shown that many natural com-
pounds alter the permeability of the cell membrane,
favoring the penetration of antibiotics (Burt, 2004). Te
interaction with bacterial enzymes can also be related
to the synergistic mechanism of natural products with
antibiotics (Wendakoon & Sakaguchi, 1995), which can
be obtained from an extract or from the combination of
extracts, synthetic products, antibiotics and other natural
products (Hemaiswarya et al., 2008; Wagner & Ulrich-
Merzenich, 2009).
Tere are many reports that condensed tannins and
gallic acid are responsible for diferent antimicrobial
activities (Ishida et al., 2006). Santos et al. (2002) reported
that the degree of polymerization is an important factor in
biological activity. Tis has been discussed by Field and
Lettinga (1992) who demonstrated that an increase in
the degree of polymerization progressively increases the
degree of reaction to tannins. Previous studies showed
that the number of hydroxyl groups in the B-ring afects
the level of growth inhibition of several microorgan-
isms, suggesting that proanthocyanidin trihydroxylated
B-rings may have better antimicrobial action. Te high
degree of polymerization and hydroxylation of the con-
densed tannins appears to be an important factor in its
antifungal activity. Scalbert proposed diferent mecha-
nisms to explain tannin antimicrobial activity. Tese
include (i) inhibition of extracellular microbial enzymes
by complexating with substrates of bacteria and fungi; (ii)
a mechanism involving the complexation of tannins with
metabolic ions required for microbial growth, and (iii)
direct action of tannins on microbial metabolism through
inhibition of oxidative phosphorylation (Scalbert, 1991).
In addition, tannins are able to complex with other mol-
ecules including macromolecules such as proteins and
polysaccharides (Haslam, 1996).
For the species S. rotundifolium, the investigation of
the modulatory activity of bark extracts represents a pio-
neering and promising study for the development of new
antimicrobial drugs. However, studies on the synergism
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Activity of Stryphnodendron rotundifolium 1269
2011 Informa Healthcare USA, Inc.
between natural products and antibacterial drugs have
been conducted with other plant species, for example,
Momordica charantia L. (Cucurbitaceae), Turnera
ulmifolia L. (Turneraceae), Hyptis martuisii Benth. and
Mentha arvenis L. (Lamiaceae) (Coutinho et al., 2009ac,
2010).
Conclusions
Te screening of active molecules in the barbatimo
extract is an important research activity that should be
considered in future studies. It is very likely that tannin
is the main antimicrobial constituent in the plant extract;
however, the exact mechanism and the compound
responsible for the antimicrobial activities are currently
unclear. Further studies need to clarify if there is a syner-
gistic efect of molecules responsible for the antimicro-
bial activity.
Acknowledgements
We are thankful to Fundao Cearense de Amparo
Pesquisa (FUNCAP-Brazil/processes 1081/06 and
0191-2.10/08 and BPI grants) and Conselho Nacional
de Desenvolvimento Cientfco e Tecnolgico (CNPq-
Brazil) for providing support to this research and to
Faculty of Medicine of Juazeiro do Norte and Faculty of
Leo Sampaio for providing animals to this research.
Declaration of interest
Te authors report no declarations of interest.
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