Short communication

Antimicrobial activity of natural products from the skins of the semiarid living
lizards Ameiva ameiva (Linnaeus, 1758) and Tropidurus hispidus (Spix, 1825)
I.J.M. Santos
a
, H.D. Melo Coutinho
b,
*
, E.F. Ferreira Matias
b
, J.G. Martins da Costa
c
, R.R. Nbrega Alves
d
,
W. de Oliveira Almeida
a
a
Laboratorio de Zoologia, Brazil
b
Laboratorio de Microbiologia e Biologia Molecular, Brazil
c
Laboratorio de Pesquisa de Produtos Naturais, Universidade Regional do Cariri, URCA, Brazil
d
Departamento de Biologia, Universidade Estadual da Paraiba, UEPB, Brazil
a r t i c l e i n f o
Article history:
Received 8 April 2011
Accepted 19 August 2011
Available online 16 September 2011
Keywords:
Antimicrobial activity
Zootherapy
Ameiva ameiva and Tropidurus hispidus
a b s t r a c t
The reptiles are the animal species most often used in traditional medicine. Tropidurus hispidus and
ameiva ameiva are two species of lizards utilized as medicines in zootherapic practice in Brazilian
semiarid region. In this work, we evaluated the antimicrobial activity of extracts of the skin of Ameiva
ameiva (MEAA) and T. hispidus (METH). The samples were tested against standard and multiresistant
strains of Escherichia coli, Staphylococus aureus and Pseudomonas aureuginosa, alone and in combination
with aminoglycoside antibiotics. Alone, none of the samples showed signicant inhibition of bacterial
growth at clinically relevant concentrations. However, combinated with the antibiotics, MEAA potenti-
ated the effect of amikacin and gentamicin, reducing their minimal inhibitory concentration (MIC) and
the growth of E. coli and S. aureus. METH lowered the MIC of gentamicin against S. aureus. Chemical
prospecting of the extracts revealed the presence of alkaloids in both, which can account for the
modulatory action of the extracts, indicating a promising source of new drugs with antibiotic properties.
2011 Elsevier Ltd. All rights reserved.
Humans have utilized animals for producing drugs and to treat
diseases and injuries since ancient times. Wild animals and their
products constitute ingredients essential in the preparation of
traditional medicines. For many years, ethnobiological studies have
been directed more to medicinal plants, but recently, various
investigators demonstrated that in culture/nature interaction there
is notable utilization of fauna for medicinal purposes in different
human societies (Alves and Rosa, 2010). In Brazil, there are reports
documenting at least 290 species are used in popular medicine.
Particularly for the Northern and Northeastern regions, various
works report that animals are widely utilized and sold for thera-
peutic purposes in both rural and urban areas (Alves et al., 2008).
Reptiles are among the animal species most often utilized in
traditional medicine. Alves et al. (2008) reported that a total of 165
species of reptiles belonging to104genera and30families are usedin
popular medicineindifferent countries of theworld. The groups with
the largest number of species utilized are snakes (60 species), fol-
lowed by lizards (51), turtles and tortoises (43) and crocodilians (11).
According to Ferreira et al. (2009, 2010), studies with extracts of
reptiles have been conducted with the aim of demonstrating their
antimicrobial and pharmacological activities. T. hispidurs and A.
Ameiva are among the species utilized in traditional medicine by
communities living in the Northeastern region, a semiarid
ecosystem, which have not yet been studied from a pharmacolog-
ical point of view.
Tropidurus hispidus, also known as the lava lizard or catenga,
lives in 14 Brazilian states, of which 9 are in the Northeast region,
and its occurrence has been recorded for the semiarid (Abreu et al.,
2002). It is found in diverse habitats, mainly on tree trunks, rocks
and walls and in open areas (heliophiles) (Abreu et al., 2002). This
lizard is diurnal and is territorial, and it is a sedentary and oppor-
tunistic forager that feeds mostly on arthropods, some plants,
basically owers, small vertebrates and needs constant sun expo-
sure and is therefore found generally in open areas (Abreu et al.,
2002). According to Alves et al. (2008), T. Hispidus (Spix, 1825), it
is used in the treatment of boils in rural areas in the semiarid
region. Besides, this species is used in the treatment of sore throat,
tonsillitis and pharyngitis. Costa Neto (2000) pointed out the
popular medicine use of Tropidurus torquatus in the treatment of
chickenpox.
* Corresponding author. Tel.: 55 8386601556.
E-mail address: hdmcoutinho@gmail.com (H.D. Melo Coutinho).
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Journal of Arid Environments
j ournal homepage: www. el sevi er. com/ l ocat e/ j ari denv
0140-1963/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jaridenv.2011.08.005
Journal of Arid Environments 76 (2012) 138e141
Ameiva ameiva (Linnaeus, 1758) is a species of lizard commonly
known as calango verde or calango and has a one of the
broadest geographical distributions among the neotropical lizards,
occurring in Brazil, it is found in most of the country, including in
the semiarid (Vanzolini et al., 1980). According to Huey and Pianka
(1981) it is considered a species with a feeding strategy of the active
foraging type, providing it was a variety of prey. According to Alves
et al. (2010), these lizards are used in traditional medicine for
inammation, dermatitis, venereal diseases, and snake bites.
Some diseases treated by communities using the traditional
knowledge report the utilization of these lizards against pathogenic
bacteria and fungi, suggesting that these species are used as
potential antimicrobial agents, which shows the need for studies
aimed at determining the medicinal efcacy of these natural
products based on these species of lizards.
The use of zootherapeutic products is also of importance from
the point of view of public health, because diseases can be trans-
mitted through their medicinal use (Alves and Rosa, 2007; Ferreira
et al., 2009). As Alves et al. (2008) have noted, the social use of the
biodiversity in countries such as Brazil is crucial for biological
conservation, public health, sustainable use of natural resources
and biological prospecting. According to Still (2003), there are few
studies assessing the true role of animal remedies, but are
extremely necessary. Knowledge about the true role of these zoo-
therapeutic agents from a chemical point of view is scarce. There-
fore, the aim of this work was to determine the antimicrobial
activity of the methanolic extract of the skin of T. hispidus and
Ameiva ameiva, alone or in combination with antibiotics.
The animals were collected (Permission for collection: 154/2007
no. 23544-1 process no. 17842812) in the municipality of Crato
(7

14
0
03
00
S 39

24
0
34
0
W), Ceara, Brazil, in April 2010. They were
caught manually, besides by active collections by rummaging
through habitats where these animals can be found. Once the
lizards were collected and sacriced, their skins were removed and
dried in a drying oven to prepare extracts. Control specimens were
xed in 70% alcohol and deposited in the zoology collection of the
Universidade Regional do Cariri/LAZ-URCA (Table 1).
The extracts of A. ameiva and T. hispidus were prepared by
submersing the skins (which were perforated to increase surface
area) for 72 h in methanol (cold extraction). Afterward, they were
ltered and concentrated using a rotary evaporator (Soxhlet)
(model Q-344B e Quimis, Brazil), and completely dried in water
bath at 70

C for 2 h (model Q-214M2 e Quimis, Brazil). The yields
for the crude extracts are given in Table 2. The extracts were then
stored in a freezer until later analyses. The experiments were
carried out with clinical isolates of Escherichia coli (EC27), Staphy-
lococcus aureus 358 (SA358) and Pseudomonas aureuginosa (PARB1),
as wells as the standard strains E. coli ATCC 10536, Staphylococcus
aureus ATCC 25923 and P. aureuginosa ATCC 15692 (Coutinho et al.,
2008). All strains were maintained on heart infusion agar (HIA,
Difco) slants, and before the assays, the cells were grown overnight
at 37

C in brain heart infusion broth (BHI, Difco). The drugs used
namely gentamicin, kanamycin, amikacin and neomycin were ob-
tained from Sigma Chemical Corp, St. Louis, MO, USA. All drugs
were dissolved in sterile water just before use.
The test solution of extracts of the two species was prepared by
dissolving 10 mg of the samples in 1 mL of dimethylsulfoxide
(DMSO- Merck, Darmstadt, Germany), obtaining an initial
concentration of 10 mg/mL; this solution was then diluted to
1024 mg/mL. The minimal inhibitory concentrations (MIC) of the
extracts were determined by microdilution assays in BHI broth with
bacterial suspensions of 10
5
CFU/mL and drug concentrations
varying from 1 mg/mL to 1024 mg/mL (in 2-fold serial dilutions)
(Javadpour et al., 1996). MIC was dened as the lowest concentra-
tion of drug at which no bacterial growth was observed. For the
evaluation of extracts for antibiotic-modifying activity, MICs of the
antibiotics were determined in the presence of each extract at sub-
inhibitory concentrations, and the plates were incubated for 24 h at
37

C.
The chemical tests to determine the presence of heterosides,
saponins, tannins, avonoids, steroids, triterpenes, cumarins,
quinones, organic acids and alkaloids were performed according to
the method described by Matos (1997). The tests are based on the
visual observation of a color change or formation of a precipitate
after the addition of specic reagents.
Methanolic extracts of the skin of A. ameiva and T. hispidus did
not showantibacterial activity at clinically relevant concentrations,
with MIC 1024 for all bacterial strains tested, thus suggesting that
the medicinal use of the lizards is ineffective against bacterial
infections.
METH and MEAA were then tested for antibacterial activity in
synergy with antibiotics of common use. When combined with the
aminoglycosides kanamycin and neomycin, the extracts did not
demonstrate any modulatory effect on antibiotic activity against
the three strains tested (EC27, SA358 and PARB1). However, when
combined with the aminoglycoside amikacin, MEAA showed
synergism, reducing the MIC of amikacin from 156.25 to 19.53
against the multiresistant strain EC27. In combination with genta-
micin, MEAA also demonstrated synergism, reducing the MIC of the
aminoglycoside from39.06 to 9.76 against the strain EC27, and with
SA358, a reduction in MIC was also observed, from 39.06 to 9.76.
The same was seen with METHplus gentamicin, where the MIC was
lowered from 39.06 to 9.76 against SA358. An antagonistic effect
was found with METH when combined with amikacin, increasing
its MIC from 39.06 to 156.25 against the strain SA358 (Table 3). The
tests for chemical prospecting showed the presence of alkaloids in
the extracts of the lizards.
The present work showed a non-clinically relevant MIC for the
extracts of T. hispidus and A. ameiva, 1024 in all cases, like other
studies that have demonstrated antimicrobial inefciency in the
medicinal use of some natural products tested. Ferreira et al. (2009)
demonstrated the ineffectiveness of the body fat of Tupinambis
merianae as an antimicrobial, a product that is used in popular
medicine for infections caused by bacteria such as E. coli and S.
aureus, also obtaining a MIC 1024 mg/mL. On the other hand,
a synergistic effect was observed with the extracts tested in
combination with particular aminoglycosides, reducing the MIC of
the antibiotics against the bacterial strains used, which has also
been found in other studies on natural products of animal as well as
plant origin. Therefore, there is a need to understand how these
substances act in order to increase the activity of conventional
antibiotics, since a substantial decrease in the concentration of
Table 1
Species utilized in the antimicrobial analyses.
Species University/Archive no.
Tropidurus hispidus Universidade Regional do Carri-URCA LZ-847
Ameiva ameiva Universidade Regional do Carri-URCA LZ-746
Table 2
Fresh weight, dry weight and yield of methanolic extracts of Tropidurus hispidus and
Ameiva ameiva (g).
Species Fresh weight Dry weight Solvent Crude extract Yield
Tropidurus hipidus
Skin 89.14 44.5275 Methanol (METH) 1.9672
Ameiva ameiva
Skin 26.00 10.3998 Methanol (MEAA) 1.303
METH- Methanol extract of Tropidurus hispidus; MEAA- Methanol extract of Ameiva
ameiva.
I.J.M. Santos et al. / Journal of Arid Environments 76 (2012) 138e141 139
aminoglycosides would be a promising improvement in the
chemotherapy of infections. According to Matias et al. (2010) the
mechanisms by which animal- or plant-derived natural products
can inhibit the growth of microorganisms differ and can be partly
due to the hydrophobic nature of some components. Various
components of the extracts can act as permeabilizers of the cell
membrane, increasing the cellular uptake of antibiotics (Helander
et al., 1998). The interference on bacterial enzyme systems can
also be a potential mechanism of action (Wendakoon and
Sakaguchi, 1995). These mechanisms of action can be involved in
the combination of an antibiotic with a natural product extract at
a sub-inhibitory concentration added directly to the culture
medium (Coutinho et al., 2010).
The presence of alkaloids in the extracts used in this antimi-
crobial assay can be a strong indication that these substances
account for the biological activity shown in the antibiotic-
modifying assay, since these extracts when combined with ami-
noglycosides signicantly reduced the MIC of the latter. Many
studies have demonstrated the pharmacological activity of alkaloids
(Paulo et al., 1992). Alkaloids with antimicrobial activity were also
demonstrated in the extract of amphibian skin (Gomes et al., 2007).
In cases where antagonism is seen between the test substances
and antibiotics, as demonstrated in the present work, it is very
important to understand the causes underlying this process, since
the combined use of antibiotics with other substances can lead to
diverse effects, positive as in the cases of synergism and negative
with antagonism, also observed in this study. According to
Granowitz and Brown (2008), an antagonistic effect due the
combined use of antibiotics probably results from a binding inter-
action. This is likely an indication of an antagonistic effect of the
interaction between METH and the aminoglycosides, reducing
antibiotic activity.
It is important to note that the use of natural products
combined with commercial drugs has been previously reported.
Calvet-Mir et al. (2008) reported the use of traditional medicine
products combined with Eastern medicine for the treatment of
diarrhea, vomiting and stomach ache. The combination of natural
products with conventional drugs can be very benecial as
demonstrated in this study, where the MIC of particular antibiotics
was signicantly reduced due their interaction with the extracts
tested. On the other hand, such combinations may pose a health
risk when the effects of natural products are not known, as could
be observed here, where the combination of METH with amikacin
resulted in a signicant decrease in antibiotic efcacy. Shin et al.
(2007) made a survey in 304 persons who had had a stroke in
the city of Kwangju in Korea, where 34% of those interviewed
acknowledged using traditional medicine combined with eastern
medicine. Alves and Rosa (2010), who studied the popular
marketing of medicinal animals in North and Northeast Brazil,
pointed out the need of considering their interaction with allo-
pathic medications (which the zootherapeutic product user can be
taking) and their possible negative effects.
According to Ferreira et al. (2009, 2010) studies of substances
from reptiles have been conducted to determine their pharma-
cological activities. The venom of the snake Bothrops jararaca
possesses various medicinal properties. Ciscotto et al. (2009)
described the antibacterial and antiparasitic activities of L-amino
acid oxidase from the venom of this snake. Products from other
species of reptiles were also studied in attempt to elucidate
their pharmacological proprieties. Liu et al. (2007) demonstrated
the anti-tumor effect of extracts of the lizard Gecko japonicus,
which is widely utilized in Chinese traditional medicine. The
lysozymes of the turtles Trionyx sinensis, Amyda cartilagenea and
Chelonia mydas demonstrated a strong antibacterial activity
(Thammasirirak et al., 2006).
The extracts of T. hispidus and A. ameiva alone did not show
clinically relevant effects against the bacterial strains tested, but in
combination with the aminoglycosides used, they mostly demon-
strated a signicant synergistic effect, whereas an antagonistic
effect with METH in combination with amikacin. A likely explana-
tion for the biological effects of these extracts is the presence of
alkaloids in the extracts of these lizards from semiarid. More
detailed studies are warranted in order to identify these alkaloids
and to determine their relation to the antibacterial activity of the
extracts of the lizards T. hispidus and A. ameiva and demonstrate the
efcacy of these natural resource used in the traditional medicine
by semiarid living communities.
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Table 3
MIC (mg/ml) of aminoglycosides in the absence and presence of 128 mg/mL of methanolic extracts of skin of T. hispidus or A. ameiva against Escherichia coli 27, Staphylococcus
aureus 358 and Pseudomonas aeuruginosa RB1.
EC 27 SA 358 PA RB1
MIC MIC combined MIC MIC combined MIC MIC combined
Antibiotic METH MEAA METH MEAA METH MEAA
Kanamycin 156.2 156.25 156.2 625 625 625 1250 1250 1250
Amikacin 156.2 156.25 19.5 39.0 156.2 39.0 625 625 625
Neomycin 78.1 78.12 78.1 156.2 156.2 156.2 1250 1250 1250
Gentamicin 39.0 39.06 9.8 39.0 9.8 9.8 312.5 312.5 312.5
METH e Methanolic extract of Tropidurus hispidus; MEAA e Methanolic extract of Ameiva ameiva.
I.J.M. Santos et al. / Journal of Arid Environments 76 (2012) 138e141 140
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I.J.M. Santos et al. / Journal of Arid Environments 76 (2012) 138e141 141