ws0">sion, in 100 mL of distilled water for 3 minutes, follow-
ing vacuum filtration and lyoph ilization.
5. Fractionation
The lyophilized aqueous crude extract was chroma-
tographed on a Sephadex LH20 column eluted with etha-
nol (absolute or 50%), methanol (absolute or 50%) and
acetone (70%) gradient solvent system. Obtained frac-
tions (186) were analyzed by thin layer chromatog-
Figure 1. Brazilian Cerrado regions in the States of São Paulo, Minas Gerais and Goiás, sites of collection of S. adstringens
ccessions. a
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Antimicrobial Activity and Rates of Tannins in Stryphnodendron adstringens
Mart. Accessions Collected in the Brazilian Cerrado 2195
raphy (TLC) and pooled according to their chemical pro-
file in 3 fractions designated I, II and III, being the last
the richer in tannin contents.
6. Antifungal Assay
A clinical Trichophyton rubrum isolate (ATCC-MY-
A3108) was obtained from a patient admitted to the
UNAERP University Hospital in Ribeirão Preto, SP,
Brazil. The mutant strain TruMDR2 was obtained from
disruption of the TruMDR2 gene from MYA3108 [12].
Standard techniques for microorganism manipulation and
growth were previously described [10]. Susceptibility of
the MYA3108 and TruMDR2 mutant strains was
tested by assessing the minimal inh ibitory concentrations
(MIC) values exhibited by different concentrations of
aqueous extract and fractions of S. adstringens diluted in
10% dimethyl sulfoxide (DMSO) according to the
M38-A microdilution technique proposed by the Na-
tional Committee for Clinical Laboratory Standards
(2002) [13]. The final concentration of DMSO, used in
the antifungal assay was fixed at a maximum of 0.5%.
Microliter trays were incubated at 28˚C and MICs were
recorded after 7 days of incubation. The MIC100 was de-
fined as the lowest concentration of the extract or frac-
tion that completely inhibited the growth of fungal
strains. The assays were carried out in three independent
experiments performed in triplicate. Fluconazole (0.07
mg/mL) was used as reference control.
7. Antibacterial Assay
Staphylococcus aureus (ATCC 6538) and Escherichia
coli (ATCC 25922) strains were used as test organisms.
Antimicrobial activity against the ATCC strain was
evaluated according to the microdilution method in a
BHI medium [14]. Briefly, each experimental well con-
taining different concentration s of crude extract and frac-
tions diluted in 10% DMSO was inoculated with the
bacterial suspension in a final concentration of 105
CFU/mL. DMSO final concentration for antifung al assay
was fixed at a maximum of 0.5%. Microdilution trays
were incubated at 37˚C and the MIC100, as defined
above, was re corded after 24 hours of incubation . MIC100
was defined as the lowest concentration of the extract or
fraction that completely inhibited the growth of bacterial
strains. The assays were carried out in three independent
experiments performed in triplicate. Ampicillin and
chloramphenicol (10 mg/mL) were used as reference
controls.
8. Tannin Evaluation
Tannin concentration was determined by modified col-
orimetric assay, absorbance wavelength at 750 nm, as
described in the Brazilian Pharmacopoeia [15] for S. ad-
stringens cortex. Powdered bark (0.75 g) was placed in a
round-bottomed flask (250 mL) containing 100 mL of
distilled water and heated for 30 min at 90˚C - 100˚C
temperature. After cooling under running water the ex-
tract was transferred to volumetric flask (250 mL) and
the volume was completed with distilled water. After
decantation, the solution was filtered through filter paper
for three times.
9. Statistical Analysis
Experiments were conducted in a Completely Random-
ized Design (CRD), and the statistical analysis of ob-
tained data was carried out using the software SISVAR,
Federal University of Lavras, MG, Brazil, applying the F
test to verify differences between treatments and the
Scott-Knot test (p > 0.05) for comparison of treatment
means. Pearson’s correlation was used to determine the
correlation between concentration of tannins and mini-
mal inhibitory concentrations (MIC).
10. Results and Discussion
MIC values determined for S. adstringens extracts indi-
cated their significant an tifungal activity. The MIC value
obtained for aqueous crude extract was 56 µg/mL against
both strains of T. rubrum. The fraction I presented higher
antifungal activity for ATCC MYA3108 mutant strain
and for TruMDR2 wild strain (312 µg/mL and 1250
µg/mL respectively).
Obtained results indicate a synergic action among
compounds present in S. adstringens crude extract that
can enhance antifungal activity (Table 1). [16], reported
similar results when testing the efficacy of S. adstringens
extracts against different strains of th e same mic roorg an-
ism.
The synergic effect of secondary metabolites found in
plant crude extracts has been continuously demonstrated.
[17] screening Plinia glomerata, a plant species as rich in
phenolic compounds as S. adstringens, reported that P.
glomerata crude extract showed more significant anti-
Table 1. MIC values (µg/mL) determined for aqueous ex-
tract and fractions from S. adstringens plants collected in
Delfinópolis-MG against T. rubrum strains.
Material T. rubrum strains
Mutant MYA3108
Crude extract 156 156
Fraction I 312 1250
Fraction II 62500 62500
Fraction III 1250 1250
Fluconazole 70 70
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Antimicrobial Activity and Rates of Tannins in Stryphnodendron adstringens
Mart. Accessions Collected in the Brazilian Cerrado
2196
fungal activity than the purified fractions or the pure
compounds isolated from that plant.
With the exception of the accessions collected in
Delfinópolis every other presented different MIC values
for each individual and dissimilarities on tannin concen-
tration were observed inter and intra plant populations.
Considering the concentration of tannin the S. adstrin-
gens accessions were divided into 2 groups: the first group
comprised individuals from Botucatu, Sacramento, Araxá,
Lagoa Formosa, Cristalina, Caldas Novas and São João
da Aliança which presented lower tannin rates (21.76%
to 28.34%) and the individuals from Paranapanema,
Cristais Paulista, Delfinópolis, Luislândia and Campo
Alegre composed the second group which exhibited
higher percentages of tannins (30.44% to 37.09%). Ac-
cordingly, all the investigated accessions presented supe-
rior rates of tannins considering the 8% minimum rec-
ommended by the Brazilian Pharmacopoeia [15].
The variation found in the accumulation of tannins in S.
adstringens accessions may be related to both genetic
diversity and edaphic characteristics. Soil collected in the
natural habitat of the investigated accessions of S. ad-
stringens was structurally different and according to [18]
soil fertility directly influences the production of tannins
in S. adstringens, being that populations native to less
fertile soil accumulate higher amount of tannins.
S. adstringens collected in Delfinópolis (MG) pre-
sented higher rates of tannins and significant inhibition
against T. rubrum strains (Table 2). Moreover it was
observed that the greater the production of tannin, the
lower was the volume of extract necessary for inhibiting
mutant strain (r2 = 0.339; Figure 2) and wild strain (r2 =
0.259; Figure 3), those results confirm that tannins play
a role on S. adstringens antifungal activity and that other
compounds like flavonols and polymeric tannins isolated
by [6] may comprise a synergic action that promote anti-
fungal activity.
[16] reported three hypothesis that might explain the
antimicrobial mechanism of tannins: inhibition of en-
zyme activity by complexation with substrates of bacteria
and fungi; direct action of tannins on the microorganism
metabolism, through the inhibition of oxidative phos-
phorylation; a mechanism involving the complexation of
tannins with metabolic ions, decreasing the availability
of essential ions to the metabolism of the microorgan-
isms.
Regarding the antimicrobial activity of S. adstringens,
the lyophilized crud extracts fro all investigated acces-
sions showed no antibacterial activity against S. aureus
(ATCC 6538) and E. coli (ATCC 25922) presenting MIC
values > 10.000 µg/mL. Similar results were reported by
[19], investigating hydroalcoholic extract from a single S.
adstringens plant against E. coli strain. However, as there
are reports on the bactericidal effect of S. adstringens
hydroalcoholic extract (96:4 v/v) against Staphylococcus
aureus (ATCC 12692), S treptococcus mitis and Lactoba-
cillus casei [16] it would be necessary additional inves-
tigations to validate S. adstringens bactericidal activity.
11. Conclusions
In this work the aqueous extract exhibited significant
activity and it was observed a positive correlation be-
tween tannin production and antimicrobial action.
Plants collected in Delfinópolis, MG, presented higher
rates of tannin and also greater inhibition against T. rubrum
Table 2. Rates of tannins and MIC values (µg\mL) obtained for S. adstringens aqueous extracts against T. rubrum strains.
MIC T. rubrum fungal strains
Collection sites % Tannin Mutant MYA3108
Araxá (MG) 21.81b 351.56a 546.87c
Botucatu (SP) 28.34b 421.87 a 499.99b
Caldas Novas (MG) 27.34b 390.62a 468.75b
Campo Alegre (GO) 30.80a 304.68a 429.68b
Cristais Paulista (SP) 30.44 a 440.62a 625.00b
Cristalina (GO) 21.76b 359.37a 4 8 4.37b
Delfinópolis (MG) 32.85a 249.98a 273.42a
Lagoa Formosa (MG ) 26.72b 888.88c 343.75a
Luislandia (MG) 37.09a 381.94a 425.34b
Paranapanema (SP) 32.20a 296.87a 578.12c
Sacramento (MG) 23.87b 507.81a 359.65a
São João da Aliança (GO) 23.80b 687.50b 765.62d
Means follow ed by the s ame letter do not statistically differ (Scott-Knott P > 0.05). Fluconazole (70 µg/mL) was use d as reference control.
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Antimicrobial Activity and Rates of Tannins in Stryphnodendron adstringens
Mart. Accessions Collected in the Brazilian Cerrado 2197
Figure 2. Correlation between tannin concentration and MIC values for S. adstringens extracts against TruMDR2 T. ru-
brum mutant strain.
Figure 3. Correlation betwe en tannin concentration and MIC values for S. adstringens extracts against MYA3108 T. rubrum
strain.
strains, indicating the superiority of genotypes native to
that region. Obtained results evid ence that S. adstringens
accessions from that population could be selected for
genetic improvement programs and used as high-quality
source material for the production of phytopharmaceuti-
cals.
12. Acknowledgements
We thank the Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP) for financial support (Process:
2008/00762-0; 2008/10499-4) and to Professor Nilce M.
Martinez-Rossi for kindly providing the T. rubrum mu-
tant strain.
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