Antimicrobial Activity and Rates of Tannins in <i>Stryphnodendron adstringens</i> Mart. Accessions Collected in the Brazilian Cerrado

doi:10.4236/ajps.2013.411272

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American Journal of Plant Sciences, 2013, 4, 2193-2198

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411272

Open Access AJPS

2193

Antimicrobial Activity and Rates of Tannins in

Stryphnodendron adstringens Mart. Accessions Collected in

the Brazilian Cerrado

Giovana Maria Lanchoti Fiori1*, Ana Lucia Fachin1, Valéria S. C. Correa2, Bianca Waleria Bertoni1,

Silvana Giuliatti3, Saulo França Amui3, Suzelei de Castro França1, Ana Maria Soares Pereira1

1University of Ribeirão Preto—UNAERP, Ribeirão Preto, Brazil; 2Reserve EcoCerrado Brazil, Araxá, Brazil; 3Department of

Genetics, University of São Paulo—USP, Ribeirão Preto, Brazil.

Email: *[email protected]

Received September 2nd, 2013; revised October 20th, 2013; accepted October 25th, 2013

bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

ABSTRACT

Inner bark extracts of Stryphnodendron adstringens, a leguminous tree species known as Barbatimão, are often incor-

porated to phytotherapic formulations due to their antimicrobial and healing activities. In this study, extracts from S.

adstringens accessions collected in 12 distinct locations were investigated in order to determine the rates of tannins in

inner barks and to validate S. adstringens antibacterial and antifungal effectiveness. Yields of tannins were quantified

by colorimetric assay following methodology described in the Brazilian Pharmacopoeia and the antimicrobial activity

was determined by microdilution technique proposed by the National Commi ttee for Clinical Laboratory Standards us-

ing S. adstringens h ydroalcoho lic and aqueous extr acts and semi-purif ied fractions. Inv estigated extracts did not present

significant antibacterial activity though aqueous extracts exhibited antifungal effect against both Trichophyton rubrum

mutant and clinical strains (MIC 156 µg/mL). A positive correlation b etween tannin concentration and antifungal activ-

ity was observed and the accessio ns collected in Delfinópolis (MG) were considered elite.

Keywords: Cerrado; Barbatimão; Genetic Diversity; Phytoterapy

1. Introduction

Stryphnodendron adstringens, a leguminous tree species

endemic to the Cerrado (savanna biome) is popularly

known as barbatimão and its inner bark extracts are tra-

ditionally used for ulcer and wound healing [1]. S. ad-

stringens extract is rich in condensed tannins which are

known to induce tissue repair [2].

Preclinical trials reported the regenerative action of S.

adstringens inner bark aqueous extracts [3] and clinical

trials validated S. adstringens healing action on pressure

ulcers [4], leading to the development of an effectively

commercialized phytopharmaceutical brand named Fito-

scar®.

Several studies using S. adstringens inner bark extracts

have reported their efficacy as anti-inflammatory in

subacute and chronic models of inflammation [5], anti-

nociceptive [6], anti-protozoal [7] and antimicrobial [8].

Human infections caused by fungi and bacteria in-

creased drastically in the last years. The treatment of

mycosis is based on the therapy used against bacteria and

antifungals, which are less prevalent if compared to an-

tibacterial agents. Additionally, the therapeutics used

against microorganisms is not always effective, causing

resistance or recurrence, producing significant toxicity.

The dermatophyte Trichophyton rubrum which usually

causes well-characterized superficial mycosis acts as

invasive in immunodepressed patients [9,10], reported

the in vitro occurrence of a mutant of the T. rubrum (gri1)

with a knock-out mutation in the ABC genes, that are

drug sensitive and can be used to test new compounds

with specific fungitoxicity [11].

Considering that the production of secondary metabo-

lites in plants and the consequen t therap eutic activity o f a

medicinal species are directly affected by its genetics,

*Corresponding a uthor.

Antimicrobial Activity and Rates of Tannins in Stryphnodendron adstringens

Mart. Accessions Collected in the Brazilian Cerrado

2194

biotic and abiotic environmental conditions and by the

interaction of all those aspects, the aim of this work was

to quantify the concentration of tannins in 12 accessions

of S. adstringens collected in 3 different states of Brazil

and determine the antimicrobial activity of extracts from

those accessions in order to identify prime regions for

collecting source material for the production of phyto-

pharmaceuticals.

2. Plant Material

Stems from S. adstringens accessions were collected in

the cities of Paranapanema, Botucatu and Cristais Pau-

lista (SP); Sacramento, Araxá, Delfinópolis, Lagoa For-

mosa and Luislândia (MG); Cristalina, Caldas Novas,

Campo Alegre and São João da Aliança (GO), totalizing

223 individuals. All collection sites were georeferenced

using Global Positioning System (GPS) (Figure 1). Inner

barks of those stems were dried in oven at 45˚C and

ground to powder (40 mesh). Flowering stems were her-

borized and 2 exsiccates of each individual were depos-

ited at the herbarium of the University of Ribeirão Preto

(Vouche r n˚ 272 - 718).

3. Hydroalcoholic Extracts

Pulverized plant material (10 g) was extracted with hy-

droalcoholic solution (100 mL - 7:3 v/v) by static mac-

eration (15 days). The extract was then evaporated and

lyophilized.

4. Aqueous Extracts

The aqueous extracts was prepared by decoction boiling

10 g of inner bark powder, of each S. adstringens acces-

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

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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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