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Natural Product Research, 2017


Evaluation of antibacterial activity of the bark and leaf

extracts of Brosimum gaudichaudii Trécul against multidrug
resistant strains
Joelma da Costa Borgesa#, Michele Cezimbra Perima#, Rodrigo Orlandini de Castrob,
Thiago Antônio de Sousa Araújoa,c, Tadeu José da Silva Peixoto Sobrinhoc,
Ana Carolina Oliveira da Silvad, Sandra Maria Botelho Marianoa,
Solange Cristina Carreiroa and Maria Cristina da Silva Prancheviciuse
Universidade Federal do Tocantins, Palmas-Tocantins, Brazil; bCell Cycle and Cancer Biology Research Program,
Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; cDepartamento de Ciências Farmacêuticas,
Universidade Federal de Pernambuco, Recife, Brazil; dHospital de Base do Distrito Federal, SMHS, Brasília,
Brazil; eDepartamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, Brazil


Brosimum gaudichaudii Trécul., a plant that belongs to Moraceae Received 1 October 2016
family, is found throughout the Brazilian Cerrado. The antimicrobial Accepted 1 March 2017
activities of ethanolic bark and leaf extracts of B. gaudichaudii were
tested against multiresistant bacteria isolated from diabetic foot Multidrug resistant bacteria;
infections (DFIs). Antimicrobial activity of the extracts was evaluated antimicrobial activity;
by agar disc diffusion (DD) and broth dilution (BD) methods. By diabetic foot infection;
BD method, bark (53.85, 45.83%) and leaf (42.31, 50.00%) extracts pharmacological potential,
contained antimicrobial activity against both gram-negative and medicinal plants
gram-positive bacteria. Increased antimicrobial activity was observed
when bark and leaf extracts were tested against Staphylococcus
aureus (63.64%) and Pseudomonas aeruginosa (66.67%). Statistical
analyses of bark and leaf extract demonstrated antimicrobial activity
against both gram-positive (p = 0.000) and gram-negative bacteria
(p = 0.012). Extract of bark (p = 0.075) or leaf (p = 0.005) associated with
ACA antibiotic showed antimicrobial activity against gram-positive
bacteria. Our study suggests that the bark and leaf extracts contain
bioactive compounds with antimicrobial activity against multidrug
resistant strains.

CONTACT  Maria Cristina da Silva Pranchevicius,

These authors contributed equally to this work and their names were arranged in alphabetical order.
 Supplemental data for this article can be accessed at
© 2017 Informa UK Limited, trading as Taylor & Francis Group
2   J. D. C. BORGES ET AL.

1. Introduction
Diabetic foot infections (DFIs) constitute a major clinical and financial burden to the diabetic
patient (Turhan et al. 2015). Antibiotic resistance is considered to be a major threat in the
treatment of DFI (Lipsky 2007). The increasing prevalence of infections caused by multidrug
resistant bacteria is a global health problem due to lack of novel classes of antibiotics
(Worthington & Melander 2013). Studies have evaluated the biodiversity and richness of
Brazilian flora which are predicted to have great pharmacological potential from the plants
that occur in the different ecosystems (de Albuquerque et al. 2007). The Brosimum gaudi-
chaudii Trécul, a plant that belongs to Moraceae family is very abundant in the Cerrado and
north-east regions of Brazil (Cunha et al. 2008). Studies with B. gaudichaudii have shown the
presence of phenolic compounds (Vieira et al. 1999) in many of the plant structures, including
the reproductive parts (Jacomassi et al. 2010; Araújo et al. 2015). Thus, agar disc diffusion
(DD) and broth dilution (BD) methods were used to investigate the antimicrobial activity of
ethanolic bark and leaf extracts of B. gaudichaudii Trécul., against 68 multidrug resistant
bacteria isolated from DFIs (Perim et al. 2015) and 6 control strains.

2.  Results and discussion

Bark extract of B. gaudichaudii Trécul. showed antimicrobial activity against all bacteria strains
using BD (48.65%) or DD (47.30%) methods. In addition, antimicrobial activity was present
until the last concentration tested (0.039 mg/mL) for the gram-negative strains (3.85%) in
BD method. Analysis of leaf extract showed higher antimicrobial activity using BD method
(47.30%) compared to DD method (24.32%) (Table S1). Different results obtained from BD
and DD methods can be attributed to the polarity of the other compounds present affecting
the diffusion of bioactive elements in low concentrations of the extracts (Klančnik et al.
2010). Although in this study the bioactive compounds have not yet been isolated, the plant
extracts contain a great variety of phytochemicals which are known to exhibit antimicrobial

activity (Trease & Evans 1989; Sanches et al. 2005). Spearman’s test (Table S2) showed a
correlation between both BD and DD methods, however only bark extract inhibited bacteria
growth up to 0.312 mg/mL. Considering B. gaudichaudii Trécul. contains a great variety of
phytoconstituents (Vieira et al. 1999; Araújo et al. 2015), our results suggest many variations
of bioactive compounds among the different parts of the plant.
A large number of both gram-positive and gram-negative multiresistant bacteria are
found in DFIs. The most common bacteria strains isolated and characterised of these lesions
are gram-positive Staphylococcus aureus, Beta-haemolytic streptococci (Lancefield group A,
B, C and G) and gram-negative Pseudomonas aeruginosa, Enterococci sp, non-fermentative
strains and members of the Enterobacteriaceae sp family (Pathare et al. 1998; EL-Tahawy
2000). In this study, significant antimicrobial activity was observed in both bark and leaf
extracts against S. aureus isolated from clinical samples (63.64%) and strains acquired from
ATCC (100%), when analysed by BD and DD methods (Table S3). Bark extract was 100%
efficient in the inhibition against Beta-haemolytic streptococci growth (BD-method), while
in leaf extract this inhibition was 50% (DD-method). Analysis of antimicrobial activity in
gram-negative strains using BD and DD methods showed that leaf extract was able to inhibit
66.67% of P. aeruginosa isolated from clinical samples. This pathogen is highly resistant to
most of broad-spectrum antibiotics (Dhanasekaran et al. 2003). Extract of the bark also inhib-
ited growth of E. coli strains isolated from clinical samples (BD-100% and DD-50%). Citrobacter
sp growth was inhibited by 50% using bark extract while no antimicrobial activity was
observed using leaf extract. Bark extract was able to inhibit 57.14% (BD-method) Proteus sp
growth isolated from clinical samples. All these clinical strains were characterised as mul-
tiresistant for most antibiotics used in ulcer treatment (Perim et al. 2015). Therefore, our
results suggest B. gaudichaudii as a potential source of antibacterial compounds to be used
in the treatment of infections caused by multidrug resistant strains.
Statistical analyses were performed to demonstrate the correlation between bark or leaf
extract and the following antibiotics: cefoxitin (CFO) against gram-negative bacteria and
amoxicillin/clavulanic acid (ACA) against gram-positive bacteria (Table S4). There was no
correlation using bark (p = 0.56; 1-sided p = 1.00) or leaf (p = 0.268; 1-sided p = 0.268) extract
associated with CFO against gram-negative strains indicating independent effects. However,
a correlation was observed in bark (p = 0.075; 1-sided p = 0.06) and also in leaf extracts
(p = 0.005; 1-sided p = 0.04) associated with ACA to inhibit gram-positive bacteria growth.
It is important to note that bark and leaf extracts when used in combination, without any
antibiotics present, inhibited both the gram-positive (p = 0.000; 1-sided p = 0.00) and
gram-negative bacteria (p = 0.014; 1-sided p = 0.012) growth indicating dependent effects.
Additive or synergistic effects are often crucial to bioactivity (Aqil et al. 2006) in plants, and
studies have demonstrated these effects between antibiotics and bioactive extracts (Adwan
& Mhanna 2008). Moreover, the results observed in this study suggest interaction among
different constituents of extracts and the potentiation of antibiotic activity against multidrug
resistant bacteria.

3. Conclusion
This study demonstrated the antimicrobial activity of bark and/or leaf extracts of B. gaudi-
chaudii Trécul suggesting the presence of natural bioactive compounds capable of treating
infections caused by multidrug resistant bacteria. However, further studies which aim at the
4   J. D. C. BORGES ET AL.

isolation and characterisation of these pharmacological compounds are needed to evaluate

its efficacy and safety as new antimicrobial agents.

Supplementary material
Experimental details relating to this paper are available online, alongside Tables S1–S3.

The authors wish to thank the SESAU-TO and Thiago de Cesaro for facilitating the development of
project, the LACEN-TO who kindly provided the control strains and Christopher Allen Ness for English
translation support.

Disclosure statement
No potential conflict of interest was reported by the authors.

This work was supported by grants from CAPES (AUX-PE-PNPD 2535/2011, Process N: 23038.007229/
2011/12)-Brazil and CNPq (Universal, Process N: 485873/2013-3)-Brazil.

Maria-Cristina da Silva Pranchevicius

Adwan G, Mhanna M. 2008. Staphylococcus aureus strains isolated from clinical specimens. Middle
East J Sci Res. 3:134–139.
de Albuquerque UP, Monteiro JM, Ramos MA, de Amorim ELC. 2007. Medicinal and magic plants from
a public market in northeastern Brazil. J Ethnopharmacol. 110:76–91.
Aqil F, Ahmad I, Owais M. 2006. Evaluation of anti-methicillin-resistant Staphylococcus aureus (MRSA)
activity and synergy of some bioactive plant extracts. J Biotechnolo. 1:1093–1102.
Araújo TAS, Peixoto Sobrinho TJS, Aguiar JS, Silva ACO, Brito FU, Silva TG, Amorim ELC, Pranchevicius
MCS. 2015. Phytochemical, antioxidant and cytotoxic analysis of Brazilian Cerrado plants: preliminary
evidence of their antitumor activity. J Med Plant Res. 9:310–319.
Cunha LCD, Paula JRD, Sá VAD, Amorim MEDP, Barros ICM, Brito LAB, Silveira N. 2008. Acute toxicity of
Brosimum gaudichaudii Trécul. Root extract in mice: determination of both approximate and median
lethal doses. Rev Bras Farmacogn. 18:532–538.
Dhanasekaran G, Sastry NG, Mohan V. 2003. Microbial pattern of soft-tissue infections in diabetic
patients in South India. Asian J Diabetol. 5:8–10.
EL-Tahawy AT. 2000. Bacteriology of diabetic foot. Saudi Med. 21:344–347.
Jacomassi E, Moscheta IS, Machado SR. 2010. Morfoanatomia e histoquímica de órgãos reprodutivos
de Brosimum gaudichaudii (Moraceae). Rev Braz Bot. 33:115–129.
Klančnik A, Piskernik S, Jeršek B, Možina SS. 2010. Evaluation of diffusion and dilution methods to
determine the antibacterial activity of plant extracts. J Microbiol Methods. 81:121–126.
Lipsky BA. 2007. Empirical therapy for diabetic foot infections: are there clinical clues to guide antibiotic
selection? Clin Microbiol Infect. 13:351–353.
Pathare NA, Bal A, Talvalkar GV, Antani DU. 1998. Diabetic foot infections: a study of microorganisms
associated with the different Wagner grades. Indian J Pathol Microbiol. 41:437–441.

Perim MC, Borges JC, Celeste SRC, Orsolin EF, Mendes RR, Mendes GO, Ferreira RL, Carreiro SC,
Pranchevicius MCS. 2015. Aerobic bacterial profile and antibiotic resistance in patients with diabetic
foot infections. Rev Soc Bras Med Trop. 48:546–554.
Sanches NR, Cortez DAG, Schiavini MS, Nakamura CV, Filho BPD. 2005. An evaluation of antibacterial
activities of Psidium guajava (L.). Braz Arch Biol Technol. 48:429–436.
Trease GE, Evans WC. 1989. Pharmacognosy. 11th ed. London, UK: Macmillan.
Turhan V, Mutluoglu M, Acar A, Hatipoglu M, Onem Y, Uzun G. 2015. Increasing incidence of Gram-
negative organisms in bacterial agents isolated from diabetic foot ulcers. J Infect Dev Ctries. 15:707–
Vieira IJC, Mathias L, Monteiro VFF, Braz-Filho R, Rodrigues-Filho E. 1999. A new coumarln from Brosimum
gaudichaudii Trecul. Nat Prod Lett. 13:47–52.
Worthington RJ, Melander C. 2013. Combination approaches to combat multi-drug resistant bacteria.
Trends Biotechnol. 31:177–184.