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Arch. Biol. Sci., Belgrade, 64 (2), 459-464, 2012 DOI:10.2298/ABS12024597S
459
ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF
LICANIA TOMENTOSA (BENTH.) FRITSCH (CRHYSOBALANACEAE)
J.B.N.F. SILVA^1 , I.R.A. MENEZES^3 , H.D.M. COUTINHO^4 , F.F.G. RODRIGUES2,5,
J.G.M. COSTA2,5^ and C.F.B. FELIPE^5
1 Universidade Federal de Pernambuco – UFPE; Departamento de Antibióticos - DANT; Laboratório de Bioensaios para
Pesquisa de Fármacos – LBPF, Recife (PE), Brazil
2 Universidade Regional do Cariri – URCA; Departamento de Química Biológica – DQB; Laboratório de Pesquisa em
Produtos Naturais – LPPN, Crato (CE), Brazil
3 Laboratório de Farmacologia e Química Molecular– LFQM, Crato (CE), Brazil
4 Laboratório de Microbiologia e Biologia Molecular– LPPN, Crato (CE), Brazil
5 Faculdade Leão Sampaio, Juazeiro do Norte, (CE), Brazil
Abstract - This work describes the chemical composition, and evaluates the antimicrobial and antioxidant activities of a
hydroalcoholic extract from the leaves of the Licania tomentosa. Gram positive and negative bacterial strains were used in
this work. Examination of the phytochemical composition of L. tomentosa revealed the presence of secondary metabolites
such as tannins, flavonoids, saponins, alkaloids, steroids and triterpenoids. An antibacterial assay pointed out that the ex-
tract had a lower minimal inhibitory concentration (MIC - 32 μg/mL) towards Staphylococcus aureus (ATCC12692). The
extract also presented antibacterial activity against other assayed bacteria, with the MIC varying between 64 and 512 μg/
mL. Our findings reveal that the extract presented an antioxidative capacity lower than that of BHT at the same concentra-
tion, used as positive control. Our results suggest that the levels and combinations between the secondary metabolites of
this plant should be investigated to explain the demonstrated antibacterial activity.
Keywords: Licania tomentosa, Chrysobalanaceae, antibacterial activity, antioxidant activity
INTRODUCTION
The use of medicinal plants to combat various dis-
eases as an alternative therapy, mainly by groups with
health assistance difficulties, is common in develop-
ing countries due its accessibility and low cost. Ac-
cording to the World Health Organization (2002), it
is important to invest in traditional medicine to im-
prove the general health status (Silveira et al., 2007).
The Chrysobalanaceae family comprises 17 gen-
era and about 450 species with worldwide distribu-
tion (Brummitt, 1992). Some species are used in folk
medicine as hypoglycemic, anti-inflammatory, and
for the treatment of diarrhea, dysentery and malar-
ia (Castilho et al., 2000; Zuque et al., 2004; Agra et
al., 2008, Ruiz-Teran et al., 2008). Other species of
Chrysobalanaceae have presented cytotoxic, antitu-
mor, antifungal, antibacterial, toxic and antioxidant
activities (Suffness et al., 1988; Braca et al., 2002; Lee
et al., 1996; Garo et al., 1997; Fernandes et al., 2003;
Zuque et al., 2004).
Licania tomentosa, typical in the northeastern
region of Brazil, is popularly known as “oiti” and
is used as a hypoglycemic and diuretic (Castilho et
460 J.B.N.F. SILVA ET AL.
al., 2000; Lorenzi, 2000; Machado et al., 2006; Ros-
sato et al., 2008). Previous studies were related mol-
luscicidal, antitumoral and antiviral activities (Bilia
et al., 2000; Miranda et al., 2002; Fernandes et al.,
Therefore, considering the potential pharmaco-
logical properties of Chrysobalanaceae species, the
objective of this work was to evaluate the antioxidant
and inhibitory activity of L. tomentosa against bacte-
rial pathogens.
MATERIALS AND METHODS
Plant material and extract preparations
Leaves of Licania tomentosa (Benth.) Fritsch, Chrys-
obalanaceae, were collected in the municipality of
Juazeiro do Norte, Ceará State, Brazil, in July 2009
(7º 12’ 47” S; 39º 18’ 55” O). The plant material was
identified and a voucher specimen was deposited
with the respective number #44569 at the Herbari-
um Prisco Bezerra, Department of Biology, Federal
University Ceara (UFC). A quantity of 280 g of aerial
parts was dried at 50ºC for 72 h. The material was
extracted by maceration using 1 L of 95% ethanol
and water (1:1) as a solvent at room temperature,
and the homogenate was allowed to stand for 72 h at
room temperature. The extract was then filtered and
concentrated under vacuum in a rotary evaporator
(model Q-344B – Quimis, Brazil) and ultrathermal
bath (model Q-214M2 – Quimis, Brazil) followed by
lyophilization until complete dehydration, resulting
in a yield of 10.87%.
Phytochemical content
Examination of the phytochemical composition of
hydroalcoholic extracts from L. tomentosa was un-
dertaken in order to detect the presence of secondary
metabolites was performed following the method
described by Matos (1997).
Antibacterial activity evaluation
The antibacterial activities of the extracts were
investigated by employing a microdilution meth-
od, recommended by NCCCLS M7-A6 (NCCLS,
2003). Brain Hear Infusion Broth (BHI 3.8%) was
used for bacterial growth (24 h, 35±2ºC). The
inoculum was an overnight culture of each bac-
terial species in BHI broth diluted in the same
media to a final concentration of approximately
1x10 8 UFC/mL (0.5 nephelometric turbidity units
(McFarland scale). After this, the suspension was
diluted to 1x10 6 UFC/mL in 10% BHI. 100 μL of
each dilution were distributed in 96-well plates
with extracts in different concentrations, achiev-
ing 5x10 5 UFC/mL as the final concentration of
the inoculum. Nine bacterial strains were used,
clinical isolates or standard strains: Staphylococ-
cus aureus MR6538, S. aureus ATCC12692, Bacil-
lus cereus ATCC33018, Pseudomonas aeruginosa
ATCC15442, Klebsiella pneumoniae ATCC10031,
Proteus vulgaris ATCC13315, Escherichia coli
MR27, E. coli ATCC25922, E. coli ATCC10536).
The extracts were dissolved in distilled water and
dimethyl sulfoxide (DMSO) to a concentration
of 1024 μg/mL. Further serial dilutions were per-
formed by the addition of BHI broth to reach a
final concentration in the range of 512 at 8μg/mL.
All experiments were performed in triplicate, and
the microdilution trays were incubated at 35±2 o^ C
for 24 h. Antibacterial activity was detected using
a colorimetric method by adding 25 μL of resa-
zurine staining (0.01%) aqueous solution in each
well at the end of the incubation period. The mini-
mal inhibitory concentration (MIC) was defined
as the lowest the extracts were able to inhibit bac-
teria growth.
Antioxidant activity
The free radical scavenging activity of the extract
Licania tomentosa plant was evaluated as described
by Mensor et al. (2001). Briefly, the plant extract was
mixed with a 0.3 mM DPPH ethanol solution, to give
final concentrations of 5, 10, 25, 50 and 125 μg of ex-
tract per ml of DPPH solution. After 30 min at room
temperature, the absorbance values were measured
at 518 nm and converted into the percentage antioxi-
dant activity.
462 J.B.N.F. SILVA ET AL.
In this study we considered EC 50 = 35.5 μg/mL
from BHT to evaluate the antioxidant potential of
natural products using different concentrations. The
antioxidant activity data obtained are shown in Table
2. The hydroalcoholic extract does not present signif-
icant activity in the decomposition of DPPH radicals
compared to BHT, a a synthetic antioxidant (EC 50 =
73.33 μg/mL).
Oxidative compounds are natural byproducts
of metabolism. However, when there are discrep-
ancies between the production of oxidizing agents
and their degradation, oxidative stress occurs. This
problem can cause cell damage and is related to a
number of diseases. Antioxidants are compounds
or substances responsible for quenching free radi-
cals (Sies, 1991; Dröge, 2002; Santos et al., 2010).
The extract from L. tomentosa showed a good anti-
oxidant effect that can be related to the presence of
polyphenols, such as ursolic acid, and flavones such
as lupeol. The activity of polyphenols against several
forms of cancer, proliferative diseases, inflammation,
and neurodegeneration is well-reported (Ciriolo et
al., 2008) and is mainly exerted through the inhibi-
tory and modulatory activities against a wide range
of receptors, enzymes and transcriptional factors
(Rice et al., 2004). Flavonoids and flavones showed
an antioxidative activity by different mechanisms,
including the scavenging of free radicals, chelation
of metals, as well as the mediation and inhibition of
enzymes. Kadam et al. (2010) have also associated
these natural products with other important effects
to health, where these products demonstrated anti-
carcinogenic and antimutagenic potentials related
to their antioxidative property, which is an impor-
tant effect resulting from the protection against cel-
lular oxidative damage. The antimicrobial activities
of tannins are also well documented. The growth of
many fungi, yeasts, bacteria and viruses was inhib-
ited by tannins.
Synthetic antioxidants such as butylated hy-
droxyanisole (BHA) and butylated hydroxytoluene
(BHT) are applied to fat and fatty foods to prevent
oxidative deterioration. However, carcinogenic and
anticarcinogenic properties have been reported
for both synthetic antioxidants (Botterwerck et al.,
2000). Therefore, there is an increasing interest in
finding antioxidants derived from natural origins to
prevent oxidative stress.
CONCLUSION
The results reported here are relevant and can be
considered as the first information about the in vitro
antimicrobial and antioxidant properties of L. to-
mentosa. Our study confirms that the extract of L. to-
mentosa presented antimicrobial and antioxidant ac-
tivities. We suggest that the data obtained here may
depend on the chemical composition of this species.
Other studies are necessary to evaluate the actions of
the isolated phytocompounds and to determine the
real effect of these natural products alone or together
to the demonstrated activities, creating options to
use this plant as a source of natural products against
infectious agents and diseases resulting from oxida-
tive damage.
Acknowledgments - The authors are grateful to the Brazilian
agencies FUNCAP, CNPq and CAPES for financial support
and FIOCRUZ for the microbial lines.
REFERENCES
Agra, M.F., Silva, K.N., Basilio, I.J.L.D., Freitas, P.F. and J.M. Bar- bosa-Filho (2008). Levantamento das plantas medicinais usadas na região Nordeste do Brasil. Rev. Bras. Farmacogn. 18, 472-508.
Alves, E.G., Vinholis, A.H.C., Casemiro, L.A., Furtado, N.A.J.C., Silva, M.L.A., Cunha, W.R. and C.H.G. Martins (2008). Estudo comparativo de técnicas de screening para aval- iação da atividade antibacteriana de extratos brutos de espécies vegetais e de substâncias puras. Quim. Nova. 31 , 1224-1229.
Aquilano, K., Baldelli, S., Rotilio, G. and M.R. Ciriolo (2008). Role of nitric oxide syntheses in Parkinson’s disease: a review on the antioxidant and anti-inflammatory activity of poly- phenols. Neurochem. Res. 33 , 2416-2426.
Bele, A.A., Jadhav, V.M. and V.J. Kadam (2010). Potential of Tannnins: A Review. Asian J. Plant Sci. 9 , 209-214.
Bilia, A.R., Braca, A., Mendez, J. and I. Morelli (2000). Mollusci- cidal and piscicidal activities of Venezuelan Chrysobalan- aceae plants. Life Sci. 66 , PL53-59.
ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OF LICANIA TOMENTOSA (BENTH.) FRITSCH 463
Bilia, A.R., Braca, A., Mendez, J. and I. Morelli (1996). Phy- tochemical investigations of Licania genus. Flavonoids from Licania pyrifolia. Pharm. Acta Helv. 71 , 199-204.
Botterwerck, A.A.M., Verhagen, H., Goldbohm, R.A., Kleinjans, J. and P.A. Brandt (2000). Intake of butylated hydroxya- nisole and butylated hydroxytoluene and stomach cancer risk: results from analyses in the Netherlands cohort study. Food Chem. Toxicol. 38 , 599-605.
Braca. A., Sortino, C., Politi, M., Morelli, I. and J. Mendez (2002). Antioxidant activity of flavonoides from Licania licaniae- flora. J. Ethnopharmacol. 79 , 379-381.
Braca, A., Bilia, A.R., Mendez, J., Pizza, C., Morelli, I. and N. de Tommasi (2003). Chemical and biological studies on Lica- nia genus. Stud. Nat. Prod. Chem. 28 , 35-67.
Brummitt, R.K. (1992). Vascular plants families and genera. Kew: Royal Botanic Garden.
Buenz, J.E., Bauer, A.B., Schnepple, D.J., Wahner-Roedler, D.L., Vandell, A.G. and C.L. Howe (2007). A randomized Phase I study of Atuna racemosa: A potential new anti-MRSA natural product extract. J. Ethnopharmacol. 114 , 371-376.
Carvalho, M.G., Candido, L.F.O., Costa, P.M., Nascimento, I.A. and R. Braz-Filho (2008). Triterpenes acids and saponins isolated from Licania arianeae Prance (Chrysobalanace- ae). J. Nat. Med. 62 , 360-361.
Carvalho, M.G. and P.M. Costa (2009). Outros constituintes iso- lados de Licania arianeae (Chrysobalanaceae). Rev. Bras. Farmacogn. 19(1B) , 290-293.
Castilho, R.O., Oliveira, R.R. and M.A.C. Kaplan (2005). Licano- lide, a new triterpene lactone from Licania tomentosa. Fi- toterapia. 76 , 562-566.
Castilho, R.O., Souza, I., Guimarães, U.P. and M.A.C. Kaplan (2000). A survey of chemistry and biological activities of Chrysobalanaceae. An. Acad. Bras. Ciênc. 72 , 289-295.
Castilho, R.O. and M.A.C. Kaplan (2008). Constituintes químicos de Licania tomentosa Benth. (Chrysobalanaceae). Quím. Nova. 31 , 66-69.
Dröge, W. (2002). Free radicals in the physiological control of cell function. Physiol. Rev. 82 , 47-95.
Fernandes, J., Castilho, R.O., Costa, M.R., Wagner-Souza, K., Ka- plan, M.A.C. and C.R. Gattass (2003). Pentacyclic triter- pens from Chrysobalanaceae species: cytotoxicity on mul- tidrug resistant and sensitive leukemia cell lines. Cancer Lett. 190 , 165-169.
Garo, E., Maillard, M., Hostettmann, K., Stoeckli-Evans, H. and S. Mavi (1997). Absolute configuration of a diterpene lactone from Parinari capensis. Helv. Chim. Acta. 80 , 538-544.
Lee, I.K-soo., Shamon, L.A., Chaia, H.B., Chagwedera, T.E., Bestlimam, Y.M., Farnswort, N.R., Cordell, G.A., Pezzuto,
J.M. and A.D. Kinghorn (1996). Cell-cycle specific cyto- toxicity mediated by rearranged ent-Kaurene diterpenoids isolated from Parinari curatelifolia. Chem. Biol. Interact. 99 , 193-204.
Lorenzi, H. (2000). Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas no Brasil. Nova Odessa: Insti- tuto Plantarum.
Machado, R.R.B., Meunier, I.M.J., Silva, J.A.A. and A.A.J.F. Cas- tro (2006). Árvores nativas para a arborização de Teresina, Piauí. Rev. SBAU. 1 , 10-18.
Matos, F.J.A. (1997). Introducao à fitoquímica experimental. For- taleza: UFC.
Mensor. L.L., F.S., Leitão, G.G., Reis, A.S., dos Santos, T.C., Coube, C.S. and S.G. Leitão (2001). Screening of Brazilian plant extracts for extracts for antioxidant for antioxidant activ- ity by the use of DPPH free radical method. Phytother. Res. 15 , 127-130.
Miranda, M.M.F.S., Gonçalvez, J.L.S., Romanos, M.T.V., Silva, F.P., Pinto, L. and M.H. Silva (2002). Anti-herpes simplex virus effect of a seed extract from the tropical plan Licania tomentosa (Benth.) Fritsch (Chrysobalanaceae). Phyto- medicine. 9 , 641-645.
NCCLS - National Committee for Clinical Laboratory Standards (2003). Methods for Dilution Antimicrobial Susceptibility Tests for bacteria that grow aerobically, Approved Standard M7-A6. Wayne: NIH.
Oberlies, N.H., Burgess, J.P., Navarro, H.A., Pinos, R.E., Soejarto, D.D., Farnsworth, N.R., Kinghorn, A.D., Wani, M.C. and M.E. Wall (2001). Bioactive constituents of the roots of Li- cania intrapetiolaris. J. Nat. Prod. 64 , 497-501.
Rossatto, D.R., Tsuboy, M.S.F. and F. Frei (2008). Arborização ur- bana na cidade de Assis-SP: uma abordagem quantitativa. Rev. SBAU. 3 , 1-16.
Rotava, R., Zanella, I., Silva, L.P., Manfron, M.P., Ceron, C.S., Al- ves, S.H., Karkow, A.K. and J.P.A. Santos (2009). Atividade antibacteriana, antioxidante e tanante de subprodutos da uva. Cienc. Rural. 39 , 941-944.
Ruiz-Téran, F., Medrano-Martínes, A. and A. Navarro-Ocaña (2008). Antioxidant and free radical scavenging activities of plant extracts used in traditional medicine in Mexico. Afr. J. Biotechnol. 7 , 1886-1893.
Santos, A.K.L., Costa, J.G.M., Menezes, I.R.A., Cansanção, I.F., Santos, K.K.A., Matias, E.F.F. and H.D.M. Coutinho (2010). Antioxidant activity of five Brazilian plants used as tradi- tional medicines and food in Brazil. Pharmacogn. Mag. 6 , 335-338.
Santos, S.C., Ferreira, F.S., Rossi-Alva, J.C. and L.G. Fernandez (2007). Atividade antimicrobiana in vitro do extrato de
