Silva et al.

BMC Complementary and Alternative Medicine 2013, 13:107

http://www.biomedcentral.com/1472-6882/13/107

RESEARCH ARTICLE Open Access

Anti-inflammatory, antioxidant, and antimicrobial

activities of Cocos nucifera var. typica
Rafaela Ribeiro Silva1, Davi Oliveira e Silva1, Humberto Rollemberg Fontes3, Celuta Sales Alviano1,
Patricia Dias Fernandes2* and Daniela Sales Alviano1

Abstract
Background: Teas from the husk fiber of Cocos nucifera are used in the folk medicine to treat arthritis and other
inflammatory processes. Some works show that some varieties have biological activities. However, one of the main
variety of the species, C. nucifera var. typica, known in Brazil as “gigante”, was not studied yet. Thus, this study
evaluates if this variety has the anti-inflammatory and antimicrobial activities already reported in other varieties.
Methods: C. nucifera aqueous crude extract (10, 50, and 100 mg/kg) and the reference drugs morphine (1 mg/kg) and
acetylsalicylic acid (100 mg/kg) were evaluated in models of inflammation (formalin-induced licking and subcutaneous
air pouch). The antioxidant activity was evaluated by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) photometric assay
and compared with those of the standards (quercetin, rutin, and ascorbic acid). The extract was also screened against
Candida albicans, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA), in the
agar diffusion method. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were
determined by the broth micro-dilution assay. Activities of combinations of the extract and antibiotics (methicillin or
vancomycin) against MRSA were evaluated using checkerboard assays.
Results: The extract significantly inhibited the time that the animals spent licking the formalin-injected paws (second
phase). The extract also inhibited the inflammatory process induced by subcutaneous carrageenan injection by
reducing cell migration, protein extravasation, and TNF-α production. Additionally, the extract showed an antioxidant
potential in vitro as good as standards in their antioxidant activity. The extract was active only against S. aureus and
MRSA. MIC and the bactericidal concentrations were identical (1,024 μg/ml). The extract and methicillin acted
synergistically against the clinical MRSA isolate, whereas an indifferent effect was detected when the extract was
combined with vancomycin.
Conclusions: The extract exhibits anti-inflammatory activity through the inhibition of the cell migration. The mixture of
extract constituents and methicillin could lead to the development of a new combination antibiotic against MRSA
infections.
Keywords: Cocos nucifera, Anti-inflammatory activity, Antioxidant activity, Antimicrobial activity

Background C. nucifera has antibacterial, antiviral [2], antioxidant [3],

The varieties of Cocos nucifera are found all over the anti-neoplastic [4,5], and anti-inflammatory activities [6].
tropical regions. In Brazil, there are different varieties like The variety “anão” is divided in “green”, “yellow” and
the “gigante” [1]. The aqueous crude extract of husk fiber “red”. The “mestiço” are known as segregated hybrids,
from C. nucifera is widely used in northeastern Brazilian resulting from aleatory cross between diverse plants, and
traditional medicine to treat diarrhea and arthritis [2]. thereby with unknown origin [1].
During the past years, it has been shown that the “comum” The inflammatory process is a complex phenomenon
from vascular tissues to harmful stimuli, such as pathogens,
damaged cells or irritants [7]. In this paper, the anti-
* Correspondence: [email protected]
2
Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas,
inflammatory action of an aqueous crude extract of husk
Laboratório de Farmacologia da Dor e da Inflamação, Rio de Janeiro, Brasil fiber from C. nucifera was assessed in two animal models.
Full list of author information is available at the end of the article

© 2013 Silva et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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Since, reactive oxygen species (ROS) have been implicated Phytochemical composition
as novel second messengers that help regulate cellular The lyophilized crude aqueous extract was submitted to
events like inflammation [8], the free radical scavenging HPLC/DAD analyses, according to a protocol devised by
properties was either evaluated. Peng et al. [13] to analyze procyanidins in grape seeds.
Besides the antioxidant and anti-inflammatory activities, Samples (20 μl) were loaded onto a 4.6 x 250 mm
the antimicrobial action was also evaluated by antibacterial reverse phase RP-18 column (250 mm X 4,6 mm X 5 μm;
and antifungal activities assays in this study, once the XTerra®). Elution was achieved with solvents A (0.02%
antimicrobial properties of the aqueous extracts of phosphoric acid in water) and B (82% acetonitrile, 0.04%
husk fiber from another Brazilian and Nigerian C. nucifera phosphoric acid) as follows: 0 to 15% solvent B in the first
varieties were previously described [2,9]. Since combination 15 min, 15 to 16% from 15 to 40 min, 16 to 17% from 40
therapies may results in the administration of a lower dose to 45 min, 17 to 43% from 45 to 48 min, 43 to 52% from
of commercial antimicrobials, which might reduce drug 48 to 49 min, held isocratic at 52% from 49 to 56 min,
toxicity and improve efficacy [10], combinations of the C. reduced from 52 to 43% from 56 to 57 min, and from 17
nucifera extract and commercial antimicrobials were also to 0% from 58 to 60 min. Peaks were detected at 280 nm.
performed in this paper. The concentration of procyanidins in the sample solution
Considering that previous studies demonstrated the of C. nucifera lyophilized crude aqueous extract was deter-
low toxicity of aqueous crude extract of husk fiber from mined by the vanillin-HCl assay [14]. Briefly, 2.5 ml of
other varieties of C. nucifera [3,6], this is also expected methanol (control) or 1% vanillin solution in methanol
for the “gigante” variety. In the popular use of this (sample) and 2.5 ml of 9 M HCl in methanol was added
plant extract, the occurrence of adverse effects is not to a test tube containing 1 ml of catechin solution (0 to
documented and aqueous crude extracts generally 300 μg/ml in methanol) or test solution (150 to 250 μg/ml
have low acute toxicities [11]. polyphenols in methanol). The reaction mixture was incu-
Furthermore, the use of the husk fiber might lead to the bated for 20 min at 30°C, and the absorbance at 500 nm
production of novel low-cost therapies and adjunct was measured. The absorbance was calculated as follows
treatments because it is a by-product from the processing for each standard and sample solution: a calibration curve
of C. nucifera [6]. Therefore, the purpose of this study was prepared using the calculated absorbance for the
was to evaluate the anti-inflammatory, antioxidant catechin solution, and the total procyanidin in each test
and antibacterial activities of a variety of C. nucifera that solution was calculated from the calibration curve.
has not been investigated yet, the “gigante”.
Animals
Materials and methods All experiments were performed with male Swiss
Plant material mice (20–25 g) obtained from our own animal facility.
C. nucifera (Arecaceae) var. typica, commonly known as Animals were maintained in temperature-controlled room
“gigante”, was collected in Sergipe, Brazil (Campo (22±2°C) with a 12 h light/dark cycles and free access to
Experimental de Itaporanga, Embrapa Tabuleiros Costeiros) food and water. Twelve hours prior to each experiment,
and authenticated by the forest engineer Humberto the animals received only water in order to avoid food
Rollemberg Fontes (Fitotecnia researcher). The charac- interfering with the substance absorption. Animal care and
terization and identification of this variety was done research protocols (ICBDFBC-015) were in accordance
following morphological, phenological, and production with the principles and guidelines adopted by the Brazilian
aspects of the plant, in addition to other criteria, College of Animal Experimentation (COBEA), approved by
such as age of the plant [12]. A voucher specimen the Ethical Committee for Animal Research (Biomedical
was deposited (number ASE- 27,441) at the Federal Science Institute/UFRJ).
University of Sergipe.
Drugs and extract administration
Preparation of C. Nucifera crude extract Acetylsalicylic acid (ASA) and morphine hydrochloride
The water extract from husk fiber was prepared by infu- were purchased from Sigma (St. Louis, MO, USA) and
sion, as described previously [2]. The use of aqueous extract Merck Inc. (Brazil), respectively. They were dissolved in
was in accordance with the popular medicinal knowledge. sterile water just before use. The extract was dissolved in
The extract was filtered, lyophilized and stored at −20oC, sterile water and administered by oral gavage at doses
yielding around 10% of the dry weight of starting material. of 10, 50, and 100 mg/kg in a final volume of 0.1 ml.
For the experiments described below, the aqueous crude Morphine (1 mg/kg) and ASA (100 mg/kg) were used
extract was re-suspended in distilled water. In the case of as reference drugs and were also administered by oral
the antimicrobial assays, it was sterilized by filtration gavage. The negative control group received vehicle
using a 0.22 μm membrane. by oral gavage.
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Formalin test Microorganisms and antimicrobial standards

The formalin test was conducted in a manner similar to Methicillin and vancomicin were obtained from Sigma-
that described by Gomes et al. [15]. Animals received an Aldrich (Brazil) and stored according to the supplier’s
injection of 20 μl of formalin (2.5% v/v) into the dorsal instructions. The following reference strains were used:
surface of the left hind paw. The time that the animal spent Staphylococcus aureus ATCC 6538 and the fungus
licking the injected paw was recorded. The response Candida albicans ATCC 36802. The study also included
consists of two phases. The first phase occurs 5 min imme- a methicillin-resistant Staphylococcus aureus strain (BMB
diately following the formalin injection (neurogenic pain 9393) and a clinical isolate of Escherichia coli obtained
response), and the second phase occurs 15–30 min after from University Hospital of the Federal University of
formalin injection (inflammatory pain response). The Rio de Janeiro.
animals were pretreated with oral doses of extract,
ASA, morphine or vehicle 60 min before the injection Antimicrobial activities
of formalin. The antimicrobial activity of the C. nucifera extract was
evaluated by the agar diffusion method described by Hili
et al. [19]. Microorganisms (2 x 105 cells) were spread over
Subcutaneous air pouch (SAP) model
appropriate plate (Brain Heart Infusion agar for bacteria
The method was similar to that described by Romano et al.
and Sabouraud agar for the fungus). The extract was
[16] with several modifications described in Raymundo
diluted in water (10 mg/ml), sterilized by filtration, and
et al. [17]. Briefly, air pouches were produced by subcuta-
10 μl aliquots of aqueous crude extract were applied to
neous injection of sterile air (10 ml) into the intrascapular
newly inoculated plates. Plates were incubated at 37°C for
region of the mice. After three days, another injection of
24 h (bacteria) or for 48 h at room temperature (fungus).
air (10 ml) was performed in order to maintain the
pouches. Three days after the last injection of air, animals
Minimal inhibitory concentration (MIC) and minimal
received an injection of sterile carrageenan suspension
bactericidal concentration (MBC) assays
(1%) into the SAP. Mice were pre-treated with oral doses
The MICs values of the extract against the test microor-
of extract, ASA or vehicle 1 h before and 23 h after carra-
ganisms were determined by broth microdilution method
geenan injection in the SAP. Animals were sacrificed 24 h
as recommended by CLSi [20]. The microdilution method
after carrageenan injection and the cavity was washed with
was also used to determine MBC values. The substances
1 ml of sterile PBS. The total number of cells was deter-
were transferred to a microplate in order to obtain two-
mined with the aid of a haemocytometer. The exudates
fold serial dilutions of the original substance. Then, an
were centrifuged at 170 x g for 10 min at 4°C, and the
inoculum (10 μl) containing 5×106 CFU/ml was added to
supernatants were collected and stored at −20°C to further
each well and the microplates were aerobically incubated
analysis. Supernatants from exudates collected in the SAP
at 37°C for 24 h. Wells without inoculum added were used
were used to measure tumor necrosis factor-α (TNF-α)
for sterility control, and the positive controls comprised of
and protein. TNF-α was quantified by enzyme-linked
inoculated growth medium without the substances.
immunosorbent assay (ELISA), using the protocol supplied
Bacterial growth was indicated by the presence of turbid-
by the manufacturer (B&D, USA). The protein content of
ity and a pellet on the well bottom, which was confirmed
each supernatant was determined using the BCA method
with 30 μl of resazurin (Sigma-Aldrich) added aseptically
(BCATM Protein Assay Kit, Pierce).
and incubated at 37°C for 1 h. The MIC was defined as the
lowest extract concentration that was able to completely
Antioxidant activity determined by the 2,2-diphenyl-1- inhibit the bacterial growth. Methicillin and vancomicin
picryl-hydrazyl-hydrate (DPPH) photometric assay were used as antimicrobial standards.
The free radical scavenging activity of the C. nucifera To evaluate whether the action of the extract was
extract was evaluated as described by Mensor et al. microbiostatic or microbicidal, 20 μl of the microbial
[18]. Briefly, the plant extract was mixed with a 0.3 culture were removed from wells with concentrations
mM 2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH) equal to, or higher than, the MIC, inoculated on BHI
ethanol solution, to give final concentrations of 0.78, agar plates, and incubated at 37°C for 24 h. The MBC
1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg of extract was defined as the lowest extract concentration at which
per ml of DPPH solution. After 30 min at room all of the bacteria have been killed.
temperature, the absorbance values were measured at
518 nm and converted into the percentage of antioxidant Synergistic activity of the extract and antimicrobial drugs
activity. Free radical scavenging activity of the extract against microorganisms
was compared with those of the quercetin, rutin, and According to Mahboubi and Bidgoli [21], synergistic
ascorbic acid. antimicrobial activity was evaluated by a checkerboard
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assay with the broth microdilution method. The extract procyanidins. This elution behavior and UV absorp-
and commercial antimicrobials were combined in tion characteristics indicate the presence of condensed
concentrations lower than their individual MIC values tannins [13].
by serial dilution in microplates. An inoculum (10 μl) In order to corroborate the presence of these tannins,
containing 5×106 CFU/ml was added to each well and the vanillin-HCl assay was performed. This test is highly
the microplates were incubated at 37°C for 24 h. The specific for procyanidins and involves acidic hydrolysis
visual bacterial growth was confirmed with 30 μl of of the condensed tannins followed by coupling of the
resazurin (Sigma-Aldrich) added aseptically and incubated flavanol units to vanillin. Polymeric procyanidins were
at 37°C for 1 h. detected at a concentration of 103.80 ± 10.5 mg/g of
Fractional inhibitory concentrations (FICs) were calcu- aqueous extract.
lated as the MIC of the combination of the extract and a
commercial antimicrobial divided by the MIC achieved Anti-inflammatory activity
when one of them was used individually. FIC index is Initially, the anti-inflammatory effect of the extract was
widely accepted for use in evaluating in vitro synergistic evaluated by the formalin-induced licking model. The
actions. It is the sum of FICs values and indicates the response consists of two phases and can evaluate both
nature of an interaction between two compounds. A FIC antinociceptive and anti-inflammatory activities. Centrally
index between 0.5 and 4.0 indicates an insignificant acting drugs, such as narcotics, are able to inhibit the two
interaction, whereas FIC index values <0.5 and >4.0 have phases equally. The first phase involves direct chemical
synergistic and antagonistic interactions, respectively. stimulation of nociceptive afferent fibers, leading to acute
neurogenic pain. However, the second phase involves
Statistical analysis inflammation in the paw and central sensitization [22,23].
In vitro experiments (antimicrobial and antioxidant As depicted in Figure 2, extract treatment did not
assays) were undertaken in triplicate sets. All animal reduce the time that the animal spent licking the formalin-
groups were composed of 6–10 mice and the results are injected paw during the first phase (neurogenic pain
presented as the mean ± S.D. Statistical significance response). However, during the second phase (inflammatory
between groups was determined by analyses of variance pain response), extract treatment induced dose-dependent
(ANOVA) followed by Bonferroni’s test. p values less inhibition, indicating an anti-inflammatory effect. Therefore,
than 0.05 (*p< 0.05) were considered significant. the extract could be acting through inhibition of the forma-
tion and/or liberation of inflammatory mediators or by
Results and discussion directly blocking its receptor. In order to evaluate how the
Phytochemical composition extract could inhibit the inflammatory reaction, another
Application of the reverse phase C18 HPLC protocol model of inflammation was performed, the subcutaneous
devised by Peng et al. [13] to the crude aqueous extract air pouch. This model was not achieved by previous studies
produced the chromatogram shown in Figure 1. The broad with aqueous extract of husk fiber from C. nucifera. In this
peak at 50–55 min belongs to a mixture of polymeric model, subcutaneous injections of air into the back result in

Figure 1 A trace of reverse phase HPLC analysis of C. nucifera lyophilized crude aqueous extract. The asterisk (*) in the figure indicates
polymeric procyanidins.
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Figure 2 Effects of the C. nucifera extract on formalin-induced licking in mice. Animals were pretreated by oral administration different
doses of the C. nucifera extract, morphine (1 mg/kg), or vehicle. The results are presented as mean ± S.D. (n = 6–10) of the time that the animals
spent licking the formalin-injected paws. Statistical significance was calculated by ANOVA followed by Bonferroni’s test. * indicates p < 0.05 when
comparing to the vehicle-treated group.

formation of a cavity similar to the synovium and the injec- the other doses of extract (50 or 100 mg/kg) significantly
tion of carrageenan in this cavity induces inflammation. suppressed the cell migration (Figure 3).
The site serves as a reservoir of cells and mediators that can A similar pattern was observed in the exudates protein
be measured in the locally accumulating fluid [24]. concentrations. An increase in the levels of protein was
Injection of carrageenan (1%) into the mice air pouches detected following carrageenan injection in the SAP. This
increased the number of leukocytes that migrated to the carrageenan-induced protein leakage was significantly
cavity when compared to the control that received PBS in inhibited by pre-treatment with the higher doses of extract
the SAP. Pre-treatment with lower dose of the extract (50 or 100 mg/kg) (Figure 4). These effects could be due
(10 mg/kg) was not able to significantly reduce the to disruption of inflammatory mediators’ formation or by
number of leukocytes. However, pre-treatment with inhibition of receptor function.
One of the mediators that play an important role in the
inflammatory process is TNF-α. It is released from

Figure 3 Effect of the C. nucifera extract in the subcutaneous Figure 4 Effect of the C. nucifera extract on protein leakage and
air pouch (SAP) model. Animals were pretreated by oral TNF-α production. Animals were pretreated by oral administration
administration with different doses of the extract 24 h and 1 h prior with different doses of the extract 24 h and 1 h prior to carrageenan
to carrageenan (1%) injection into the SAP. The results are presented (1%) injection into the subcutaneous air pouch (SAP). The results are
as mean ± S.D. (n = 6–10) of total leukocytes (×106/ml). Statistical presented as mean ± S.D. (n = 6–10) of protein (μg/ml) or TNF-α (pg/ml).
significance was calculated by ANOVA followed by Bonferroni’s test. Statistical significance was calculated by ANOVA followed by Bonferroni’s
* indicates p < 0.05 when comparing C. nucifera-treated mice with test. * indicates p < 0.05 when comparing C. nucifera-treated mice with
the vehicle-treated group; # indicates p < 0.05 when comparing the vehicle treated group; # indicates p < 0.05 when comparing
vehicle-treated mice to the PBS-treated group. vehicle-treated mice with the PBS-treated group.
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Table 2 Minimal inhibitory concentrations (MICs) of C.

nucifera extract and antimicrobial drugs by the micro-
dilution method
MIC* (μg/mL)
Microorganism
extract MET VCM
S. aureus 1024 0,25 nd
MRSA 1024 512 1
* All MICs were microbicidal; MET - methicillin; VCM - vancomycin; nd - not
determined.

direct antioxidant activity of flavonoids in vivo is

likely to be limited to tissues in which relatively high
concentrations of these compounds could be achieved, like
in the digestive tract. In lower concentrations, they could
Figure 5 Free radical scavenging properties of the C. nucifera support indirect antioxidant actions, like regulation of
extract, using the DPPH photometric assay. The results are pro-oxidant enzymes and inhibition of protein–receptor
presented as mean ± S.D. from three independent assays. EC50 coupling that initiates oxidant production [26].
values were determined. However, the antioxidant activity can be related to an
anti-inflammatory effect. Free radicals can attract inflamma-
tory mediators contributing to a generalized inflammatory
activated monocytes and macrophages and increases response [27]. The reactive oxygen species are linked
vascular endothelial permeability [25]. The production of to increased endothelial permeability and leukocyte
this mediator was dramatically increased by the injection transendothelial migration [28]. This can explain the
of carrageenan in the SAP. Nevertheless, the levels of this inhibition of the carrageenan-induced protein leakage
mediator in the exudates were significantly reduced in and the leukocyte migration by the extract of C.
mice that received pre-treatment with the highest dose of nucifera in the SAP.
extract (100 mg/kg) (Figure 4).

In vitro antioxidant activity Antimicrobial activity

The antioxidant activity was determined by the 2,2- The results of the agar diffusion assay showed that the
diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay. The extract does not have an antimicrobial effect against the
EC50 values were calculated, and the result obtained fungus and the Gram-negative bacterium tested. However,
for the C. nucifera extract was comparable with those an inhibition zone was observed when the extract was
of the standards (quercetin, rutin, and ascorbic acid) tested against S. aureus and MRSA (Table 1).
(Figure 5). Esquenazi et al. [2] found that the “olho de cravo” C.
A good antioxidant effect of the aqueous crude nucifera has activity against one S. aureus ATCC strain
extract of husk fiber from “olho de cravo” C. nucifera and three clinical isolates of S. aureus. Further,
has already been determined by the same model Akinyele et al. [9] studied the aqueous extracts of the
(EC50= 10.0 ± 0.7 μg/ml). This activity could be husk fiber from a C. nucifera variety from Nigeria.
attributed to the presence of condensed tannins in They demonstrated that it also has activity against S.
the husk fiber from C. nucifera [11]. aureus, in addition to inhibiting Gram-negative bacteria,
Nevertheless, the extrapolation of an antioxidant such as Escherichia coli.
effect in vitro to an in vivo action is not trivial. A In accordance to the lack of reported activity of C.
nucifera against Candida albicans in the literature, the
Table 1 Antimicrobial screening of C. nucifera extract by
agar diffusion method Table 3 Effects of combinations of antimicrobial drugs
Microorganism Antimicrobial screening
and C. nucifera extract against MRSA
MIC of drug in MIC of extract in FIC of FIC of FIC
C. albicans -
combination combination drug extract index
E. coli -
VCM 0,9375 256 0,9375 0,25 1,19 (I)
S. aureus +
MET 96 256 0,1875 0,25 0,44 (S)
MRSA +
VCM - vancomicin; MET - methicillin; MIC - minimal inhibitory concentration
(-) no activity; (+) activity. (μg/ml); FIC -fractional inhibitory concentration; I- indifferent; S – synergistic.
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“gigante” variety does not have effect against this fungus. Nevertheless, the MIC value of methicillin against
Despite of being the same species of coconut, the different MRSA was lowered from 512 to 96 μg/ml when the
varieties have different types and quantities of substances extract was added at a concentration of 256 μg/ml
in the fiber. The biological activity can be affected by (4-fold reduction in MIC). This combination had a
structural variations, such as stereochemistry [29]. FIC index calculated as 0.44, corresponding to a syn-
This may be the reason why the “gigante” variety ergistic effect (Table 3). However, this synergistic
does not have the same effects of the variety from effect was not able to reverse the methicillin resistance
Nigeria described by Akinyele et al. [9]. In the (MIC remained > 4 μg/ml).
present paper, the extract does not possess growth The extract can be fractionated in order to check if
inhibitory activity against Escherichia coli. Nevertheless, it the activity improved. In addition, the active substances
is important to note that the tested strain is different in the extract and their modes of action should be
from that of the other study. Discrepancy in findings further investigated. Moreover, the extract can lead to
may be the result of the pathogen strains utilized or the development of novel inexpensive phytomedicines or
also be due to the variety of the plant and extraction models for synthetic substances.
process used. The antistaphylococcal effect of the extract is important
Others varieties have activity against S. aureus and the because MRSA and vancomycin-intermediate S. aureus
“gigante” variety is likewise active. It was also investigated if are spreading worldwide and the pharmaceutical arsenal
it would be able to inhibit the MRSA growth and it was. available to control antimicrobial-resistant bacteria is
Thus, the minimal inhibitory concentrations of the limited. Invasive infections by S. aureus are associated with
extract against those bacteria (S. aureus and MRSA) significant morbidity and mortality, creating urgency in the
were determined and results are shown in Table 2. development of new therapies and adjunct treatments that
MIC was the same for the two bacteria strains tested act against the resistant strains [33,34].
(1024 μg/ml) and at this concentration, a microbicidal
effect was observed. MIC values were also determined
Conclusions
for methicillin. Methicillin-resistant staphylococcal is
Concerning the crude nature of the extract, the results
defined as MIC ≥ 4 μg/ml [30]. To the methicillin-resistant
allow concluding that it exhibited significant bioactivity
strain, vancomicin was used as standard antimicrobial.
and properties that support its uses in the folk medicine.
Despite having been able to killing the two strains of
The extract exhibits anti-inflammatory activity through
bacteria, the concentration of the extract was higher
the inhibition of the cell migration. In addition, the
than those of the standards antimicrobial used. How-
mixture of extract constituents and methicillin could
ever, the MIC must be interpreted in the appropriate
lead to the development of a new combination antibiotic
context. The raw extract is a mixture of active and
against MRSA infections.
non-active compounds and therefore higher MICs are
Although the modes of action remain to be completely
expected [31].
elucidated, the extract and its constituents might be
Cos et al. [32] proposed an endpoint with concentration
promising candidates for the direct or adjunct treatment
required to produce 50% growth inhibition below
of inflammation and infection by MRSA, with low cost.
100 μg/ml for extracts. Nevertheless, in the present
study it was evaluated the extract’s concentration that
Competing interests
was able to completely inhibit the bacterial growth The authors declare that they have no competing interests.
and not only 50% growth inhibition. Thus, a higher
MIC is acceptable. Webster et al. [31] interpreted a
Authors’ contributions
MIC value of crude plant extracts approximately to RRS carried out all the experiments and wrote the manuscript; DOS prepared
1000 μg/ml as showing strong antifungal potential. and characterized the C. nucifera extracts; HRF identificated the C. nucifera
The antimicrobial activity may be enhanced by variety, collected the seeds and extracted the fibers; CSA participated in the
design of the study and revised it critically for important intellectual content;
synergistic effect of natural products and antimicro- PDF participated in the design of the pharmacological study, helped to draft
bial drugs. Therefore, the checkerboard assay was the manuscript and revised it; DAS participated in the design and
performed to evaluate the antimicrobial effects of coordination of the microbiological study. All authors read and approved the
final manuscript.
combinations of antimicrobial drugs and the extract
against MRSA. When the extract was combined with
Acknowledgements
vancomycin, the MIC of the extract against MRSA We would like to thank Renata S. Zardo (M.Sc.) for assistance in SAP model.
reduced 4-fold, however the MIC of vancomycin Authors would like to thank Mr. Mark English for the English revision of the
against MRSA was almost the same. In this combination, manuscript. The present work was supported by Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional
the FIC index was 1.19, corresponding to an indifferent de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de
effect (Table 3). Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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Author details 17. Raymundo LJ, Guilhon CC, Alviano DS, Matheus ME, Antoniolli AR,
1
Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Cavalcanti SC, Alves PB, Alviano CS, Fernandes PD: Characterization of the
Góes, Laboratório de Estruturas de Superfície de Micro-organismos, Instituto anti-inflammatory and antinociceptive activities of the Hyptis pectinata
de Microbiologia Paulo de Góes, Rio de Janiero, Brasil. 2Universidade Federal (L.) Poit essential oil. J Ethnopharmacol 2011, 134:725–732.
do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de 18. Mensor LL, Menezes FS, Leitão GG, Reis AS, Dos Santos TC, Coube CS, Leitão
Farmacologia da Dor e da Inflamação, Rio de Janeiro, Brasil. 3Embrapa SG: Screening of Brazilian plant extracts for antioxidant activity by the
Tabuleiros Costeiros, Av. Beira Mar 3250 Caixa postal 44. Cep 49015-040, use of DPPH free radical method. Phytother Res 2001, 15:127–130.
Bairro Treze de Julho, Aracaju, Sergipe, Brasil. 19. Hili P, Evans CS, Veness RG: Antimicrobial action of essential oils: the
effect of dimethylsulphoxide on the activity of cinnamon oil. Lett App
Received: 1 November 2012 Accepted: 8 May 2013 Microbiol 1997, 24:269–275.
Published: 16 May 2013 20. Clinical and Laboratory Standards Institute (CLSi): Methods for Dilution
Antimicrobial Susceptibility Tests, fourth ed. Wayne, PA, USA; 2008. Approved
Standard, M7-A4.
References 21. Mahboubi M, Bidgoli FG: Antistaphylococcal activity of Zataria multiflora
1. Adebayo J, Santana A, Krettli A: Evaluation of the antiplasmodial and essential oil and its synergy with vancomycin. Phytomedicine 2010,
cytotoxicity potentials of husk fiber extracts from Cocos nucifera, a 17:548–550.
medicinal plant used in Nigeria to treat human malaria. Hum ExpToxicol 22. Ocvirk R, Murphy P, Franklin KB, Abbott FV: Antinociceptive profile of ring
2012, 31:244–249. A-reduced progesterone metabolites in the formalin test. Pain 2008,
2. Esquenazi D, Wigg MD, Miranda MM, Rodrigues HM, Tostes JB, Rozental S, 138:402–409.
Da Silva AJ, Alviano CS: Antimicrobial and antiviral activities of 23. Shibata M, Ohkubo T, Takahashi H, Inoki R: Modified formalin test:
polyphenolics from Cocos nucifera Linn. (Palmae) husk fiber extract. characteristic biphasic pain response. Pain 1989, 38:347–352.
Res Microbiol 2002, 153:647–652. 24. Shin S, Jeon JH, Park D, Jang JY, Joo SS, Hwang BY, Choe SY, Kim Y:
3. Alviano DS, Rodrigues KF, Leitão SG, Rodrigues ML, Matheus ME, Fernandes Anti-inflammatory effects of an ethanol extract of Angelica gigas in a
PD, Antoniolli AR, Alviano CS: Antinociceptive and free radical scavenging carrageenan-air pouch inflammation model. Exp Animals 2009,
activities of Cocos nucifera L. (Palmae) husk fiber aqueous extract. 58:431–436.
J Ethnopharmacol 2004, 92:269–273. 25. Mehta D, Malik AB: Signaling Mechanisms Regulating Endothelial
4. Kirszberg C, Esquenazi D, Alviano CS, Rumjanek VM: The effect of a Permeability. Physiol Rev 2006, 86:279–367.
catechin-rich extract of Cocos nucifera on lymphocytes proliferation. 26. Fraga CG, Oteiza PI: Dietary flavonoids: Role of (-)-epicatechin and related
Phytother Res 2003, 17:1054–1058. procyanidins in cell signaling. Free Rad Biol Med 2011, 51:813–823.
5. Koschek PR, Alviano DS, Alviano CS, Gattass CR: The husk fiber of Cocos 27. García-Lafuente A, Guillamón E, Villares A, Rostagno MA, Martínez JA:
nucifera L. (Palmae) is a source of anti-neoplastic activity. Braz J Med Biol Flavonoids as anti-inflammatory agents: implications in cancer and
Res 2007, 40:1339–1343. cardiovascular disease. Inflam Res 2009, 58:537–552.
6. Rinaldi S, Silva DO, Bello F, Alviano CS, Alviano DS, Matheus ME, Fernandes 28. Wittchen ES: Endothelial signaling in paracellular and transcellular
PD: Characterization of the antinociceptive and anti-inflammatory leukocyte transmigration. Front Biosc 2009, 14:2522–2545.
activities from Cocos nucifera L. (Palmae). J Ethnopharmacol 2009, 29. Kusuda M, Inada K, Ogawa TO, Yoshida T, Shiota S, Tsuchiya T, Hatano T:
122:541–546. Polyphenolic constituent structures of Zanthoxylum piperitum fruit and
7. Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE: Chronic the antibacterial effects of its polymeric procyanidin on methicillin-
inflammation: importance of NOD2 and NALP3 in interleukin-1beta resistant Staphylococcus aureus. Biosc Biotechnol Biochem 2006,
generation. Clin ExpImmunol 2007, 147:227–235. 70:1423–1431.
30. Clinical and Laboratory Standards Institute (CLSi): Performance standards for
8. Hordijk PL: Endothelial signaling events during leukocyte transmigration.
antimicrobial susceptibility testing, 18th informational supplement. Wayne, PA,
FEBS J 2006, 273:4408–4415.
USA: CLSI; 2008. document M100-S18.
9. Akinyele TA, Okoh OO, Akinpelu DA, Okoh AI: In vitro antibacterial
31. Webster D, Taschereau P, Belland RJ, Sand C, Rennie RP: Antifungal activity
properties of crude aqueous and n-hexane extracts of the husk of Cocos
of medicinal plant extracts; preliminary screening studies.
nucifera. Molecules 2011, 16:2135–2145.
J Ethnopharmacol 2008, 115:140–146.
10. Lin RD, Chin YP, Hou WC, Lee MH: The effects of antibiotics combined
32. Cos P, Vlietinck AJ, Berghe DV, Maes L: Anti-infective potential of natural
with natural polyphenols against clinical methicillin-resistant
products: how to develop a stronger in vitro ‘proof-of-concept’.
Staphylococcus aureus (MRSA). PlantaMedica 2008, 74:840–846.
J Ethnopharmacol 2006, 106:290–302.
11. Alviano WS, Alviano DS, Diniz CG, Antoniolli AR, Alviano CS, Farias LM,
33. Van Bambeke F, Mingeot-Leclercq MP, Struelens MJ, Tulkens PM: The
Carvalho MA, Souza MM, Bolognese AM: In vitro antioxidant potential of
bacterial envelope as a target for novel anti-MRSA antibiotics. Trends in
medicinal plant extracts and their activities against oral bacteria based
PharmacolSc 2008, 29:124–134.
on Brazilian folk medicine. Arch Oral Biol 2008, 53:545–552.
34. Zampini IC, Cuello S, Alberto MR, Ordoñez RM, D’ Almeida R, Solorzano E,
12. Aragão WM, Tupinambá EA, Angelo PCS, Ribeiro FE: Seleção de cultivares
Isla MI: Antimicrobial activity of selected plant species from “the
de coqueiro para diferentes ecossistemas do Brasil. In Recursos Genéticos
Argentine Puna” against sensitive and multi-resistant bacteria.
e Melhoramento de Plantas para o Nordeste Brasileiro (online). Edited by
J Ethnopharmacol 2009, 24:499–505.
Queiroz MA, Goedert CO, Ramos SSR. Petrolina: Embrapa Semi-Árido/
Embrapa Recursos Genéticos e Biotecnologia; 1999.
doi:10.1186/1472-6882-13-107
13. Peng Z, Hayasaka Y, Iland PG, Sefton M, Høj P, Waters EJ: Quantitative
Cite this article as: Silva et al.: Anti-inflammatory, antioxidant, and
analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera)
antimicrobial activities of Cocos nucifera var. typica. BMC Complementary
seeds by reverse phase high-performance liquid chromatography. J Agric and Alternative Medicine 2013 13:107.
Food Chem 2001, 49:26–31.
14. Nakamura Y, Tsuji S, Tonogai Y: Analysis of Proanthocyanidins in Grape
Seed Extracts, Health Foods and Grape Seed Oils. J Heal Sci 2003,
49:45–54.
15. Gomes NM, Rezende CM, Fontes SP, Matheus ME, Pinto AC, Fernandes PD:
Characterization of the antinociceptive and anti-inflammatory activities
of fractions obtained from Copaifera multijuga Hayne. J Ethnopharmacol
2010, 128:177–183.
16. Romano M, Faggioni R, Sironi M, Sacco S, Echtenacher B, Di Santo E,
Salmona M, Ghezzi P: Carrageenan-induced acute inflammation in the
mouse air pouch synovial model. Role of tumour necrosis factor. Med
Inflam 1997, 6:32–38.